The mAB 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope.

PROM1 (CD133, AC133) is a protein that is required for the maintenance of primary cilia. Mutation in the Prom1 gene in humans and animal models are associated with several forms of retinal degeneration. mAB 13A4 is the main reagent used to detect the mouse PROM1 protein. We endeavored to map the epitope of the rat monoclonal antibody mAB 13A4 to the mouse PROM1 protein. Deletion mutagenesis demonstrated that mAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1. Furthermore, the affinity of mAB 13A4 to the major PROM1 isoform in photoreceptor cells is significantly reduced due to the inclusion of a photoreceptor-specific alternative exon in the third extracellular domain. Interestingly, a deletion in the photoreceptor specific isoform of six amino acids adjacent to the alternative exon restored the affinity of mAB 13A4 to PROM1. The results of the mutagenesis are consistent with the computationally predicted helical bundle structure of PROM1 and point to the utility of mAB 13A4 for evaluating the effect of mutations on the PROM1 structure. Our results show that the PROM1 isoform composition needs to be considered when interpreting tissue and developmental expression data produced by mAB 13A4.

Introduction

Prominin-1 (PROM1, CD133, AC133) was identified as the antigen of monoclonal antibodies raised against human hematopoietic progenitors and mouse neuroepithelial cells [1-3]. PROM1 is a glycosylated membrane protein with five transmembrane and three extracellular domains [1, 2]. PROM1 is a member of a conserved family of proteins involved in modulating the architecture of cellular protrusions, such as microvilli and cilia [4-6]. Mutations in the human PROM1 gene have been reported in cases of Retinitis Pigmentosa, Stargardt Disease, Macular Dystrophy, Leber Congenital Amaurosis and are primarily associated with cone-rod dystrophy [7-11]. The forms of retinal degeneration associated with PROM1 mutations represent a diverse spectrum in terms of disease onset and severity of the presented symptoms. Animal models lacking Prom1 or expressing the dominant Arg373Cys mutant recapitulate the retinal degeneration phenotype and display defects in disk morphogenesis [9, 12, 13]. Most mutations in Prom1 are recessive and result in loss of function due to premature stop codons. Notably, three missense mutations, Leu245Pro, Arg373Cys, and Asp829Asn, have dominant inheritance patterns [7]. The mechanisms underlying the pathology of PROM1 mutations and the reasons for the confinement of their phenotypes to the visual system are still unclear. Recent evidence of genetic interaction with ABCA4 points to a role in transport of all-trans retinaldehyde across the photoreceptor disk membranes [14].

The Prom1 gene produces multiple splicing isoforms that can be tissue, and cell type specific [15-19]. Six alternative exons in the mouse Prom1 can potentially produce 24 splice variants, although to date only eight have been enumerated [15, 16]. In mouse photoreceptor cells a microexon introduces 6 amino acids in the photoreceptor-specific s8 isoform of Prom1 (Fig 1A) [16, 17]. This exon, referred to as exon 19 to denote its position in the coding region of the reference clone BC028286, is located at chr5:44,012,499–44,012,517 in the GRCm39 assembly of the mouse genome [15]. While exon 19 is present in most vertebrate clades, it is not used in the primates, including humans, due to mutations that disrupt either the 3’ or the 5’ splice site of the exon (Fig 1B).

Fig 1

Photoreceptor specific splice variant of PROM1.

A) Alignment of the photoreceptor-specific s8 isoform of PROM1 (RefSeq NP_001157057) to the ubiquitously expressed isoform s2 (RefSeq NP_001157049). The amino acids encoded by the photoreceptor specific exon 19 are shown in bold and shaded in gray. The third extracellular domain is underlined. B) Alignment of vertebrate exon 19 sequences including the adjacent 3’ and 5’ splice sites. Mutations inactivating the splice sites in primates are shaded in gray.

Despite playing conserved and critical functions the proteins encoded by the Prominin-1 genes show relatively low conservation of their primary amino acid sequence. For example, human and mouse PROM1 proteins share approximately 61% sequence identity. Consequently, antibodies to PROM1 tend to be species specific. In mice, the rat monoclonal antibody mAB 13A4 is the reagent typically used to detect PROM1. The mAB 13A4 antibody was raised against extract from mouse neuroepithelium [2]. Its antigen was cloned by screening for reactivity with mAb 13A4 of a phage library of mouse kidney cDNA [2]. mAB 13A4 is speculated to recognize part of the third extracellular domain of PROM1 because of a truncating mutation in that domain that abolishes its binding, but the epitope was never mapped [8, 20].

Prompted by a discrepancy between the PROM1 protein levels measured in the postnatal retina by mAB 13A4 and the mouse monoclonal antibody to PROM1 ab27699, we set out to map the epitope of mAB 13A4. We found that mAB 13A4 recognizes a structural epitope that can be disrupted by deletions in the second and third extracellular domains. Furthermore, the affinity of mAB 13A4 to PROM1 is dramatically reduced by the inclusion of the photoreceptor-specific exon 19.

Materials and methods

Animals

The Prom1 mice were acquired from the Jackson laboratory (B6. BXD83-Prom1rd19/Boc, Stock No: 026803). All experiments were conducted with the approval of the Institutional Animal Care and Use Committee at West Virginia University.

Prom1 clones

We obtained a full length Mammalian Gene Collection clone of the mouse photoreceptor specific isoform (s8) of Prom1 from Horizon Discovery (clone ID: 4502359, NCBI accession: BC028286). Gibson assembly (NEB# E5510) was used to generate Flag-tagged Prom1 clones and deletion mutants in pcDNA3.1+ (Invitrogen). All clones are deposited at AddGene (https://www.addgene.org/Peter_Stoilov/). The primers used for cloning are listed in S1 Table.

Cell culture and transfection

Mouse Neuro-2a (N2a) neuroblastoma cells (ATCC CCL-131) were cultured in OptiMEM reduced serum media buffered with sodium bicarbonate and supplemented with 4% (v/v) fetal bovine serum (FBS, R&D Systems, Minneapolis, MN). The cells were grown at 37°C in a 5% CO2 humidified atmosphere. The cDNA clones were transiently transfected in N2a cells using polyethylenimine [21]. Cell lysates for Western blot analysis were collected at 24 hours post-transfection.

Denaturing gel electrophoresis and western blot

Flash-frozen mouse retinas and N2a cells transiently transfected with Prom1-expressing constructs were lysed in RIPA buffer (50 mM Tris HCl-PH 8.0, 150 mM NaCl, 1.0% TritonX-100, 0.5% Sodium Deoxycholate, 0.1% SDS) supplied with protease (Sigma-Aldrich catalog# 535140-1ML) and phosphatase inhibitors cocktail (Sigma-Aldrich catalog # P5726-1 ML). After homogenization, the lysate was incubated on ice for 10 mins, then cleared by centrifugation for 15 mins. Where applicable glycosylation was removed by treating with deglycosylation MixII (NEB# P6044) following the manufacturer’s recommendation. 20 μg of protein extract was resolved in 4–20% polyacrylamide SDS–PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Immunobilon-FL, Millipore). After blocking with BSA in PBST (Phosphate- buffered saline with 0.1% Tween-20), the membranes were probed with primary antibodies overnight at 4°C, followed by incubation with fluorescently labeled (Alexa Fluor 647 or 488, Jackson ImmnuoResearch) secondary antibodies for 1 hour at room temperature. The membranes were then imaged on Amersham Typhoon Phosphorimager (GE Healthcare).

Serial dilution was performed to ascertain the linearity of western blot quantification (S1 Fig). Lysates of cells transfected with the s8(-ex19) clone were diluted with extracts from cells transfected with an empty vector to maintain equal loading. The lysates were then probed on western blot by mAB 13A4 and imaged as described above. Linear regression was performed in Graphpad Prism on the scaled and normalized band intensities.

Blue Native Polyacrylamide Gel Electrophoresis (BN PAGE)

The samples were lysed in BN PAGE sample buffer (Thermo Fisher Catalog# BN2008) containing 1% digitonin and protease inhibitors following the manufacturer’s recommendation. The lysates were treated with benzonase at room temperature for 30 minutes to shear the DNA, and cleared by centrifugation for 15 mins. Prior to electrophoresis, the samples were mixed with Coomassie G-250 and resolved in 3–12% NativePAGE Bis-Tris gel (Invitrogen Catalog #BN1003BOX) as per the manufacturer’s recommendations. The gels were then transferred on PVDF membranes (Immunobilon-FL, Millipore) following the manufacturer’s recommendations. After transfer, the membranes were incubated in 8% acetic acid for 15 minutes to fix the proteins, rinsed with deionized water, and air-dried. The lane containing the size standard was cut from the membrane and stained with Ponceau S, and imaged on Amersham Typhoon Phosphorimager (GE Healthcare) using the Cy2 filter set. The membranes were blocked, probed with antibodies, and imaged as described above in the denaturing gel electrophoresis protocol.

Western blot quantification

Western blot images were analyzed using ImageQuant TL (version 8.1, GE Healthcare). Band intensities in denaturing gel blots were measured after subtracting the background using the rolling ball algorithm built in the ImageQuantTL software. To enable comparison across blots, the band intensities were scaled to the total signal intensity for each blot. The signal was then normalized to the scaled intensities of the loading control.

In native gels, applying the rolling ball background subtraction algorithm and measuring individual band intensities proved unreliable due to smearing. Instead we used the signal from an adjacent lane containing extract from cells transfected with an empty vector to determine the background and quantified the total signal intensity above 240 Kda within each well. The data was then processed as described above for the denaturing gels.

Antibodies

The primary and secondary antibodies that were used throughout our studies include the following: mouse anti-β-tubulin (1:5000; catalog # T8328-200ul, Sigma-Aldrich, St. Louis, MO), mouse anti-GAPDH (1:20,000; custom made), mouse anti-flag M2 (1:1000, catalog # F1804-200UG, Sigma-Aldrich, St. Louis, MO), rat anti- Prom1(1:1000, clone ID:13A4, ThermoFisher, Waltham, MA), mouse anti-GFP HRP conjugated GFP tag (1:1000, Cat #HRP-66002, ThermoFisher, Waltham, MA), mouse anti-Prom1 (1:1000, Cat #ab27699, Abcam, Cambridge, MA), Alexa Fluor 647 conjugated AffiniPure Goat-Anti rabbit IgG (1:3000, Jackson ImmunoReserach, West Grove, PA), Alexa Fluor 488 conjugated AffiniPure Goat-Anti mouse IgG (1:3000, Jackson ImmunoReserach, West Grove, PA), and Alexa Fluor 647 conjugated AffiniPure Goat-Anti rat IgG (1:3000, Jackson ImmunoReserach, West Grove, PA).

Statistical analysis

The two-way analysis of variance (ANOVA) with Tukey Honest Significant Differences (HSD) post-hoc test or two-tailed unpaired Student’s T-test were used to determine statistical significance as indicated in the results section. Quantitative data is presented as the mean of three biological replicates ±standard error of the mean. Biological replicates refer to cell lines samples grown and transfected independently, and to tissue samples collected from different animals.

Protein structure prediction and visualization

The RobeTTa structure prediction service was used to create models of the photoreceptor-specific s8 (RefSeq NP_001157057) isoform that contains exon 19 and the ubiquitously expressed isoform s2 (RefSeq NP_001157049) that lacks exon 19 (see Fig 1A for alignment of the two sequences). To create images of the structures we used Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

Results

Discrepancy in the PROM1 protein levels measured by the mAB 13A4 and ab27699 antibodies

While investigating the expression of PROM1 in postnatal mouse retina we noticed that when measured by mAB 13A4 the PROM1 protein levels peaked at postnatal day 8, five days before the peak recorded by ab27699 (Figs 2A and 2B and S2). Furthermore, mAB 13A4 showed approximately three to five fold lower levels of PROM1 at postnatal days 13 and beyond compared to ab27699 (Fig 2A and 2B). Two way ANOVA showed significant effect of the day after birth (F(9) = 55.26, p-value<2*10−16) and the antibody used (F(1) = 365.48, p-value<2*10−16) on the measured PROM1 protein levels. According to the Tukey HSD post-hoc test the signals detected by mAB 13A4 and ab27699 were significantly different starting from postnatal day 13. It is possible that cross-reactivity of ab27699 to other proteins of size similar to PROM1 in the retina could have compromised its performance. To rule out cross-reactivity we probed retinal extracts from wild type, heterozygous, and homozygous Prom1 mutant retina with mAB13A4 and ab27699. The Prom1 allele contains a premature stop codon that abolishes the expression of the PROM1 protein [22]. Both antibodies recognized a protein just over 100KDa in size in the wild type retina that was not detected in the extract from Prom1 knockout retina (Fig 2C). Thus, the discrepancy in the signal between mAB13A4 and ab27699 is likely due to differences in the availability of the epitopes recognized by the two antibodies.

Fig 2

Discrepancy in the levels of PROM1 as determined by the mAB 13A4 ab27699 antibodies.

A) Immunoblotting for PROM1 in mouse retina lysate collected between postnatal day 0 (P0) and postnatal day 110 (P110) using the mAB 13A4 and the ab27699 antibodies. Anti-β-tubulin serves as a loading control. B) Quantification of Prom1 level in retina lysates using the mAB 13A4 and the ab27699 antibodies. Error bars represent the standard error of the mean (n = 3). Two-way ANOVA was used to assess the effect of the postnatal day and the antibody used on the PROM1 signal. The statistical significance of the signal mAB 13A4 compared to ab27699 for each day was calculated by Tukey HSD post-hoc test. Tukey HSD p-values of less than 0.001 from the post-hoc test are indicated by “***“. C) Test of the specificity of mAB 13A4 and ab27699 antibodies for detecting PROM1 using the retinal lysate from wild type mice and Prom1 mutants that do not express PROM1 protein. Arrow indicates the position of the expected PROM1 band.

Reduced affinity of mAB13A4 to PROM1 carrying the photoreceptor-specific exon 19

A short 18nt microexon, exon 19, is included in the Prom1 transcripts specifically in photoreceptor cells (Fig 1) [17]. The inclusion rate of exon 19 starts to increase at postnatal day 3 and the exon 19 containing transcripts become dominant in the retina after postnatal day 8 [17]. The peptide encoded by exon 19 is inserted in the third extracellular loop of PROM1 (Fig 3A), which is also the proposed location of the mAB 13A4 epitope [8, 20]. Thus, it was possible that inclusion of exon 19 disrupts the epitope of mAB 13A4 while leaving intact the epitope of ab27699. To determine if exon 19 disrupts the mAB 13A4 epitope we generated Flag-tagged cDNA clones that either contain (s8) or skip (s8(-Ex19)) the photoreceptor specific exon 19. The cDNA were transfected in N2a cells and their expression was probed with mAB 13A4, ab27699 and anti-Flag antibodies. As expected, mAB 13A4 showed significant reduction in its affinity to the protein containing exon 19 compared to the epitope tag (Figs 3B and 3C and S3). The inclusion of exon 19 had no effect on the affinity of the ab27699. To rule out that mAB 13A4 recognizes a glycosylated epitope we treated lysates from N2a cells transfected with s8 and s8(-Ex19) expression constructs with PGNase F before probing them by western blot. Consistent with Weigmann et al. the deglycosylated proteins were recognized by the mAB 13A4 antibody (Fig 3D) [2]. The overall signal produced by mAB 13A4 was reduced when deglycosylated PROM1 was probed resulting in inconsistent detection of the s8 isoform (Figs 3D and S4).

Fig 3

The affinity of mAB 13A4 to PROM1 is affected by alternative splicing.

A) Schematic representation of the PROM1 structure showing the position of exon 19 (black) in the third extracellular domain (EC 3) of the photoreceptor specific PROM1 isoform s8 (Adapted from Corbeil et al [23]). Extracellular domains one through three are labeled as EC 1, EC 2, and EC 3 respectively. B) Western blot of recombinant s8 and s8 lacking exon 19, s8(-Ex19), expressed in N2a cells with mAB 13A4, ab27699 and antibody to the Flag epitope. Transfection with empty pcDNA3.1 vector (EV) was used as a negative control. All transfections were spiked with vector expressing GFP to control for transfection efficiency. C) Quantification of mAB 13A4 and ab27699 signals relative to the signal from the Flag-tag antibody. Error bars represent the standard error of the mean (n = 3). Unpaired Student’s t-test was used to assess the statistical significance and the p-value is indicated on the chart. D) Deglycosylated PROM1 isoform s8 is recognized by mAB 13A4 with reduced affinity compared to protein lacking the amino acids encoded by exon 19.

mAB 13A4 recognizes a structural epitope

To map the epitope of mAB13A4 we created a series of tiled deletion mutants of the s8(-Ex19) cDNA, that originated at the point where exon 19 would have been inserted and progressed in C-terminal and N-terminal direction (Fig 4A). The deletion mutants covered 108 amino acids of sequence. Nine out of ten deletions resulted in complete loss of the mAB 13A4 epitope (Figs 4B and S4). Only the most C-terminal deletion in the series (D+4) could be detected by mAB 13A4. As with the full length protein the D+4 mutant was recognized by mAB 13A4 after being deglycosylated (S4 Fig). The results of the deletion mutagenesis indicate an epitope for mAB 13A4 that is at least 94 amino acids in length. This length far exceeds the size range of linear peptide epitopes and strongly argues for a structural rather than a linear epitope [24, 25].

Fig 4

Mapping of the mAB 13A4 epitope.

A) Sequence of the third extracellular domain. Underlining shows the positions of the deletions used for epitope mapping. The deletion variants were generated starting from the s8 clone lacking exon 19, s8(-Ex19). Solid triangle above the sequence shows the position where exon 19 is inserted in the photoreceptor specific isoform. B) Western blot analysis of PROM1 deletion mutants expressed in N2a cells using mAB 13A4 and Flag-tag antibodies. All mutants with exception of D+4 resulted in loss of the mAB 13A4 epitope. Transfection with empty pcDNA3.1 vector (EV) was used as a negative control. All transfections were spiked with a vector expressing GFP to control for transfection efficiency. C) Native gel Western blot analysis using mAB 13A4 and Flag-tag antibodies of N2a cell lysate transfected with either an empty vector (EV), s8, or s8(-Ex19). D) Quantification of mAB 13A4 signals relative to the signal from the Flag-tag antibody in native gel electrophoresis western blot. Dots represent individual data points. Line and error bars the mean and the standard error of the mean (n = 5). Unpaired Student’s t-test was used to assess the statistical significance and the p-values were less than 0.01 (indicated by “**”).

The western blots shown on Figs 1, 3 and 4 were performed using denaturing gel electrophoresis. Detecting a structural antigen using this approach would require the protein to renature on the membrane prior to probing with the primary antibody. Consequently, our results may reflect the propensity of the splicing isoforms and deletion mutants to renature rather than the genuine affinity of the mAB 13A4 to the native proteins. To test if this is the case we resolved the proteins produced by the s8 and s8(-Ex19) clones on native gel electrophoresis and probed the membranes with mAB 13A4 and anti-Flag antibodies. The exon 19 containing protein was recognized with reduced affinity (Figs 4C and 4D and S5) demonstrating that inclusion of exon 19 alters the structure of PROM1. The reduction in the mAB13A4 reactivity towards the s8 isoform on native gel was 3 fold compared to 8 fold reduction when denaturing gel was used to resolve the proteins. This difference likely reflects inefficient refolding of the PROM1 protein containing exon 19, Interestingly, under native conditions PROM1 formed higher order complexes.

Computationally derived PROM1 tertiary structure predicts the effect of sequence manipulation on the mAB13A4 epitope

To better understand the nature of the mAB 13A4 epitope and how our deletion mutagenesis affected it we needed a tertiary structure for PROM1. There are no empirically derived structures of PROM1. Nevertheless, recent advances in computational approaches for structure prediction are producing remarkably accurate structures [26, 27]. We used the RobeTTa structure prediction service to model the structures of the mouse PROM1 isoforms s8 and s2 (Fig 1A) [26]. The photoreceptor-specific s8 isoform differs from canonical s2 isoforms by the inclusion of exon 19 in the third extracellular domain and the skipping of two exons in the cytoplasmic C-terminal domain. The structures predicted by RobeTTa for the s8 and s2 proteins were in excellent agreement with each other and with the structure of the human PROM1 predicted by Alpha Fold [28]. In the predicted PROM1 structure, the second and third extracellular domain each form two antiparallel alpha helices that are continuous with the adjacent transmembrane domains. The alpha helices formed by the second and third extracellular domains and the adjacent transmembrane domains are packed in a four helix bundle (Figs 5B and S6). Inclusion of exon 19 lengthens the second helix of the third extracellular domain causing a kink in the bundle (Figs 5B and S6). Mapping the positions of the deletions that disrupted the mAB 13A4 epitope on the PROM1 structure showed that they were located towards the middle portion and the tip of the helical bundle. The D+4 mutation, which was the only one that did not result in loss of the mAB 13A4 epitope, was the furthest from the tip of the helical bundle. Based on the positions of the deleted segments and the six amino acids encoded by exon 19 in the PROM1 structure we made three predictions: (i) deleting six amino acids adjacent to exon 19 (Del AA 6) in the photoreceptor-specific PROM1 isoform should shorten the helix compensating the inclusion of exon 19, and restore the mAB 13A4 epitope (Fig 5B, colored dark blue on the structure of s8); (ii) Deletion of a 15 amino acid segment (D-7) in the third extracellular domain opposite to D+4, should retain the mAB 13A4 epitope due to its distance from the location of the epitope in the upper half of the helical bundle (Fig 5B, colored red on the structure of s2); (iii) Deletion of a 15 amino acid segment (Del EC 2) in the upper half of the second extracellular domain of s8(-Ex19) should result in loss of the mAB 13A4 epitope (Fig 5B, colored sky blue on the structure of s2). The predicted structures of the Del AA 6 and D-7 mutants lacked the kink at the tip of the helix bundle that was present in the PROM1 s8 protein isoform (S5 Fig). The Del EC 2 mutant grossly distorted the helix bundle (S6 Fig). All three predictions proved to be accurate when the proteins expressed from the corresponding clones were analyzed by Western blot (Fig 5C). Deletion of the six amino acid long segment recovered the mAB 13A4 epitope in the photoreceptor specific s8 isoform. Conversely, the deletion in the second extracellular domain of the protein encoded by the s8(-Ex19) clone resulted in complete loss of the epitope. Finally, deletion D-7 in s8(-Ex19) preserved the epitope, although the affinity of mAB 13A4 was reduced. The results of the structure directed mutagenesis provide further support for mAB 13A4 recognizing a structural epitope. In addition, our results demonstrate the utility of the modeled PROM1 structures.

Fig 5

Computationally derived tertiary structure of PROM1 predicts the effect of mutations on the mAB 13A4 epitope.

A) Sequence segments from the PROM1 protein with the amino acids deleted in clones Del EC2, D-7 and Del AA6 underlined. B) Partial structure of PROM1 isoforms s8 and s2 showing the positions of the segments deleted in the experiments on Fig 4 and on panel C of this Fig on the tertiary structure of PROM1. Exon 19 is shown in red on the structure of s8. The deletion Del AA6 analyzed on panel C is shown in blue on the structure of s8. On the structure of s2 cyan color indicates deletions D-1 through D-6 and orange indicates deletions D+1 through D+4 from the experiments shown on Fig 4. Also on the structure of s2, the positions of the deletions D-7 and EC2 analyzed on panel C are shown in red and sky blue, respectively. Arrow points to the position of the excluded exon 19 in s2. C) Western blot analysis of PROM1 deletion mutants expressed in N2a cells using mAB 13A4 and Flag-tag antibodies. All transfections were spiked with a vector expressing GFP to control for transfection efficiency.

Discussion

To reliably interpret results from techniques that employ antibodies it is essential to know the antibody epitope, its specificity and its affinity. While mapping the antibody epitope may be important, it is usually not considered necessary as long as specificity to the target can be demonstrated. Such practice leaves a gap that in certain cases can have significant impact on interpreting experimental results as we show here for mAB 13A4. mAB 13A4 is widely used to detect the mouse PROM1 protein because of its excellent specificity and the lack of alternatives with comparable performance. As of the time of writing of this article, there are over 300 publications in Google Scholar citing the 13A4 antibody in the context of PROM1. Here we show that the mAB 13A4 antibody recognizes a structural epitope and its affinity for naturally occuring PROM1 isoforms can vary dramatically. When used to measure PROM1 levels in the retina, mAB 13A4 underestimated the protein amount by a factor of five compared to ab27699. In our hands deglycosylation of PROM1 reduced the signal produced by mAB 13A4 making the detection on western blot of the unglycosylated s8 isoform unreliable. It remains to be determined if a sugar moiety contributes to the epitope of the antibody or lack of glycosylation results in inefficient refolding of the protein.

Interestingly, the reactivity of the AC133 antibody, a reagent commonly used to detect the human PROM1 protein, is reduced during cancer stem cell differentiation without corresponding loss of the protein [29]. This loss of reactivity is proposed to be due to masking of the epitope by either changes in the PROM1 structure or by PROM1 protein interactors. How often do antibodies recognize structural antigens? Barlow et al. estimate that less than 10% of antigens are continuous and most antigens are formed by residues that are brought in proximity to each other by the protein structure [30]. At the time of writing of this manuscript the Immune Epitope Database (www.iedb.org) lists 6070 discontinuous and 202950 linear peptide B-cell epitopes [31]. The prediction by Barlow et al. is not necessarily in contradiction with the Immune Epitope Database records as many of the linear peptides are likely to form a secondary structure and the epitopes may in fact be discontinuous. Furthermore, the convenience of synthetic peptides or protein fragments as immunogenes is biasing against structural epitopes. Both mAB 13A4 and AC133 antibodies were raised against full length native proteins in the context of whole cell extracts or intact cells.

To determine the exact mAB1 13A4 epitope unequivocally will require determining the structure of the PROM1—mAB 13A4 complex, which is beyond the scope of the current work. Nevertheless, we show that mAB 13A4 is a useful reagent for detecting perturbation in PROM1 structure as changes to the PROM1 sequence that were hundreds of amino acids apart abolished the mAB 13A4 epitope. Furthermore we created three mutations in PROM1 guided by the computational model of its structure. The effect of these mutations on the mAB 13A4 epitope was in line with the predicted structure, providing empirical evidence for its validity. Finally, we demonstrate that under native conditions PROM1 can form higher order complexes. Further research will be needed to determine the composition of these complexes. If the complexes prove to be PROM1 oligomers they can provide a possible path towards understanding the dominance of Prom1 mutations in patients with cone-rod dystrophy.

Primer sequences.

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Linearity of western blot quantification.

A) Western blot replicates. Serial dilution of extract expressing clone s8(-Ex19) with extract from cells transfected with an empty vector was probed by mAB 13A4. B) Plot showing scaled normalized signal intensities for each replicate and linear regression with 95% confidence interval. R2 = 0.93, p-value = 1.8*10−12.

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Gel images of replicates related to Fig 1A and 1B.

Boxes denote the parts of the images used in preparing Fig 1A.

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Gel images of replicates related to Fig 3B and 3C.

Boxes denote the parts of the images used in preparing Fig 3B.

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Deglycosylation of PROM1 deletion mutants.

Lysates from N2a cells were treated with deglycosylation mix II (NEB) and analyzed on western blot next to untreated controls. The blots were probed with mAB 13A4 and anti-Flag antibodies as indicated.

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Native gel electrophoresis of PROM1 clones s8 and s8(-Ex19).

The proteins were resolved by native blue electrophoresis, transferred to PVDF membranes, and probed with mAB 13A4 and anti-Flag antibodies as indicated. Lanes containing the size standard were cut from the membranes after the transfer and stained with Ponceau S. The size standard lanes and probed membranes were imaged on Typhoon Phosphorimager (GE Healthcare).

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Protein structure predictions for PROM1 isoform s8 and deletion clones.

Extracellular domains 2 (EC2) and 3 (EC3) are indicated on the predicted structures by green and pink color respectively. Exon 19 and the amino acids deleted in Del AA6 are colored on the structure of the s8 isoform in red and blue, respectively. The deletions for clones Del EC2 and D-7 are colored on the structure of s8(-Ex19) in light blue and red, respectively. In structures that do not contain exon 19 arrows indicate the position of the junction between exons 18 and 20. The structures of s8 and Del EC2 have pronounced kinks near the top of the bundle when compared to the structures of s8(-Ex19). Del AA6, and Del EC2.

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28 Jul 2022

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1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Matalkah et al. investigated the character of the 13A antibody, which recognizes CD133/Prom1. They found that the 13A4 recognises the structure of the Prom1 protein, rather than a single continuous sequence. Particularly the Prom1 conveying the exon 19 reduces the affinity to the 13A4 antibody. They conclude that the isofom composition needs to be considered in using this antibody because some of the isoforms may not be detected by this antibody.

The analyses have been overall done well, and provide a good piece of information for usage of the CD133/Prom1 antibody. I would support the publication of this manuscript. I have a few comments.

(1) p20. Line 4 tertiary for PROM1; Do the authors mean “tertiary structure”?

(2) Is it possible to predict the ratio of the different isoforms of Prom1 by using two different antibodies of ab27699 and m13A4? Can the authors discuss based on the data of Fig 2B?

Reviewer #2: Prom1 is a factor with an important role in the maintenance of syria and is involved in retinal degeneration; several splicing variants of Prom1 exist and are thought to have cell type-dependent regulatory mechanisms, such as being strongly expressed in neurons and other cells. In this paper, we compare mAB13A4 and ab27699 and find that the recognition efficiency differs during the post-developmental phase, indicating differential induction of splicing variants after development. They also claim that mAB13A4 recognizes two extracellular domains based on Western Blot (+SDS) validation.

While these results may have important implications for the analysis of Prom1 expression, there are several details that are of concern.

It is interesting to note that this occurs in signals that are recognized by antibodies in the post-developmental phase (Fig2). However, this phenomenon is a common one that can be observed in almost all genes with splicing variants. Furthermore, many of the antibodies sold by companies do not reveal the details of the recognition sites, and for these reasons, those conducting research with antibodies are always aware of the effects of splicing variants. Therefore, it is necessary to clarify the biological significance of this phenomenon, which can be approached by examining the following two details.

1. to clarify whether the difference in signal between mAB 13A4 and ab27699 detected in this study is due to the difference in splicing variants, it is necessary to examine mRNAs from P0 to P110 by PCR or other means. This would also allow us to further discuss the recognition of Exon19 (KFVRVR) by mAB 13A4, which is discussed in Fig. 3 and beyond.

2. it will be important to determine if the proteins detected in mAB 13A4 and ab27699 have any biological significance to maintain the significance of this paper. If the sequence of the splicing variant recognized by mAB 13A4 and ab27699 is revealed, e.g., by method 1, it should be shown experimentally.

In the description of Fig. 4, it says "Interestingly, under native conditions PROM1 formed higher order complexes corresponding in size to dimers and tetramers. Is this someone else's report? Or is this the content of Fig4C? You should clarify which one it is.

However, if you are referring to Fig. 4C, this data alone does not prove that it is a dimer. To show that it is a dimer or a tetramer, we need Immunoprecipitation, analysis using mutants of the amino acid sequence necessary for Prom1-Prom1 interaction, or analysis of the crystal structure of Prom1. For immunoprecipitation, for example, co-transfection of Flag-Prom1 and HE-Prom1, Immunoprecipitation with Flag and WB with HE. The data in Fig. 4C alone does not prove that Prom1 forms homodimers with each other, and the presence of heterodimers such as Prom1-X is also suspected.

The results of Fig5C are of great concern.

Western Blot (+SDS) would be expected to alter the structure of many of the proteins, especially membrane proteins, and not maintain the structure of a second or third extracellular domain. Therefore, Native-PAGE may alleviate this concern.

This paper uses 13A4 and ab27699, what is the difference between them?

13A4 is the hybridoma clone number and ab27699 is the product model number. 13A4 was purchased from ThermoFisher, so it seems correct to use the product model number for 13A4 as well. Even if ab27699 did not have a clone number, it would be misleading not to refer to 13A4 by the product model number. Or is 13A4 using a special purification method?

Although this paper focuses on 13A4, are the results obtained in ab27699 consistent with previous reports or information disclosed by the company? This should be included in Discussion.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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23 Aug 2022

Reviewer #1: Matalkah et al. investigated the character of the 13A antibody, which recognizes CD133/Prom1. They found that the 13A4 recognises the structure of the Prom1 protein, rather than a single continuous sequence. Particularly the Prom1 conveying the exon 19 reduces the affinity to the 13A4 antibody. They conclude that the isofom composition needs to be considered in using this antibody because some of the isoforms may not be detected by this antibody.

The analyses have been overall done well and provide a good piece of Information for usage of the CD133/Prom1 antibody. I would support the publication of this manuscript. I have a few comments.

# We thank the reviewer for the supportive evaluation of our work. Below are our responses to the specific comments.

(1) p20. Line 4 tertiary for PROM1; Do the authors mean "tertiary structure"?

# Thank you for catching this. The sentence is now fixed and reads "... a tertiary structure of PROM1" (page 14, line 297).

(2) Is it possible to predict the ratio of the different isoforms of Prom1 by using two different antibodies of ab27699 and m13A4? Can the authors discuss based on the data of Fig 2B?

# As shown in Figure 2 the signals from the ab27699 and mAb 13A4 antibodies can be used to compare two or more samples and determine if there is a difference in the isoform expression. While after careful calibration it may be possible to use the antibodies for measuring the isoform ratio we think such determination will be semi-quantitative at best. The reasons for that are:

The antibodies are not isoform specific. mAB 13A4 still recognizes both isoforms albeit it has reduced affinity to one of the isoforms. The second antibody, ab27699, recognizes both isoforms with similar affinity.

We have not exhaustively determined how post-synthetic modifications like glycosylation affect the affinity of the two antibodies. .

For quantitative assessment of isoform expression on protein level we recommend using isobaric labeling mass-spectroscopy. For the benefit of the reviewer we attach below a plot representing peptide level signals for Prom1 from a mass-spectrometry data set we recently generated (10.6019/PXD030748) in the PDF version of our response.

Reviewer #2: Prom1 is a factor with an important role in the maintenance of syria and is involved in retinal degeneration; several splicing variants of Prom1 exist and are thought to have cell type-dependent regulatory mechanisms, such as being strongly expressed in neurons and other cells. In this paper, we compare mAB13A4 and ab27699 and find that the recognition efficiency differs during the post-developmental phase, indicating differential induction of splicing variants after development. They also claim that mAB13A4 recognizes two extracellular domains based on Western Blot (+SDS) validation.

While these results may have important implications for the analysis of Prom1 expression, there are several details that are of concern.

It is interesting to note that this occurs in signals that are recognized by antibodies in the post-developmental phase (Fig2). However, this phenomenon is a common one that can be observed in almost all genes with splicing variants. Furthermore, many of the antibodies sold by companies do not reveal the details of the recognition sites, and for these reasons, those conducting research with antibodies are always aware of the effects of splicing variants. Therefore, it is necessary to clarify the biological significance of this phenomenon, which can be approached by examining the following two details.

# We thank the reviewer for carefully reviewing our work. We agree that ascribing a biological function to the alternative isoforms of Prom1 would broaden the appeal of our work. Unfortunately we do not think inclusion of exon 19 is likely to have an effect on the function of Prom1, because of its loss in primates (Figure 1). The significance of our work is that it describes an undocumented behaviour of a widely used reagent. While we agree that the potential of alternative splicing to change the antigenic properties of proteins should be routinely considered by researchers, we find little evidence in the literature that this is the case. In the case of mAB 13A4 this phenomenon remained unnoticed for 25 years after the initial publication, despite the relatively broad use of the antibody. Below are our responses to the specific comments.

1. to clarify whether the difference in signal between mAB 13A4 and ab27699 detected in this study is due to the difference in splicing variants, it is necessary to examine mRNAs from P0 to P110 by PCR or other means. This would also allow us to further discuss the recognition of Exon19 (KFVRVR) by mAB 13A4, which is discussed in Fig. 3 and beyond.

# The switch in splicing of Prom1 in the postnatal retina was shown previously by us. We would like to direct the reviewer to Murphy et al., PLoS genetics 2016, Figure 3 B where we have analyzed the splicing of Prom1 exon 19 in the retina by RT-PCR between postnatal day 0 and day 16. A copy of the figure is included in the PDF version of our response. Inclusion levels of exon 19 switch from mostly skipped at postnatal day 0 to near complete inclusion by postnatal day 12-16. Inclusion levels remain constant after postnatal day 16 based on RNASeq data from the same article and multiple RNASeq datasets published by others. The work by Murphy et al is referenced in this context on page 11 lines 216-219 in the results section.

2. it will be important to determine if the proteins detected in mAB 13A4 and ab27699 have any biological significance to maintain the significance of this paper. If the sequence of the splicing variant recognized by mAB 13A4 and ab27699 is revealed, e.g., by method 1, it should be shown experimentally.

# Prom1 has a well established role in stem cells, cancer development and vision. As we review in the introduction mutations in Prom1 are commonly associated with retinal degenerations. We do not think inclusion or skipping of exon 19 has an effect on the function of the Prom1 protein, because the exon is lost in primates (Figure 1B). While the biological significance of Prom1 isoforms containing exon 19 is questionable, our work is significant technically because of the wide use of the mAB 13A4 antibody in cancer and vision research. mAB 13A4 is the most often used reagent for detecting the mouse Prom1 protein with over 300 publications using it. We believe that it is important for investigators to know that the affinity of the antibody is affected by alternative splicing, especially when it is being used for quantitative assessment of Prom1 levels across tissues or developmental time points.

3. In the description of Fig. 4, it says "Interestingly, under native conditions PROM1 formed higher order complexes corresponding in size to dimers and tetramers. Is this someone else's report? Or is this the content of Fig4C? You should clarify which one it is.

However, if you are referring to Fig. 4C, this data alone does not prove that it is a dimer. To show that it is a dimer or a tetramer, we need Immunoprecipitation, analysis using mutants of the amino acid sequence necessary for Prom1-Prom1 interaction, or analysis of the crystal structure of Prom1. For immunoprecipitation, for example, co-transfection of Flag-Prom1 and HE-Prom1, Immunoprecipitation with Flag and W.B. with HE. The data in Fig. 4C alone does not prove that Prom1 forms homodimers with each other, and the presence of heterodimers such as Prom1-X is also suspected.

# We agree with the reviewer that the data does not prove that these bands are dimers or tetramers. We removed the reference to dimers and tetramers in this sentence (page 14 line 292) and in the Discussion section (page 18, lines 393-394). The two sentences now only point to the existence of higher order complexes without speculating about their composition.

4. The results of Fig5C are of great concern. Western Blot (+SDS) would be expected to alter the structure of many of the proteins, especially membrane proteins, and not maintain the structure of a second or third extracellular domain. Therefore, Native-PAGE may alleviate this concern.

# The reviewer is correct that SDS will denature the protein. We disagree with the reviewer that this is a problem for Figure 5. On the blot shown on Figure 5 we have two samples that show gain of signal relative to the parental s8 isoform: s8(-Ex19) and Del AA6 mutant. Thus, loss of signal due to denaturation of the proteins with SDS is not an issue. The reason is that Prom1 renatures as SDS is removed during the transfer and/or blocking of the membrane. This is not an uncommon phenomenon.

5. This paper uses 13A4 and ab27699, what is the difference between them? 13A4 is the hybridoma clone number and ab27699 is the product model number. 13A4 was purchased from ThermoFisher, so it seems correct to use the product model number for 13A4 as well. Even if ab27699 did not have a clone number, it would be misleading not to refer to 13A4 by the product model number. Or is 13A4 using a special purification method?

# The 13A4 is how this antibody was first described and how it is commonly referred to in the literature. The mAB 13A4 name is also how the antibody is recorded in the CiteAB database. In addition, the 13A4 antibody is also available from Sigma-Aldrich under a different part number. Consequently, it will be confusing if we use the vendor part number. In contrast, ab27699 is only available from AbCam and has no other designations.

6. Although this paper focuses on 13A4, are the results obtained in ab27699 consistent with previous reports or Information disclosed by the company? This should be included in Discussion.

# The antibody is consistent with the vendor’s specifications. We confirmed the specificity of the antibody using a mouse model that does not express Prom1 protein (Figure 2C). As can be seen from the figures in our manuscript the ab27699 antibody produces a higher background compared to mAB 13A4, at least in our hands. In the literature we could only find use of the antibody for IHC/IF. We cannot comment on the performance of the antibody in these applications.

Submitted filename: Response to reviewer.pdf

Click here for additional data file.

8 Sep 2022

The mAB 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope

PONE-D-22-16255R1

Dear Dr. Stoilov,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Reviewer #2: Prom1 is a factor with an important role in the maintenance of syria and is involved in retinal degeneration; several splicing variants of Prom1 exist and are thought to have cell type-dependent regulatory mechanisms, such as being strongly expressed in neurons and other cells. In this paper, we compare mAB13A4 and ab27699 and find that the recognition efficiency differs during the post-developmental phase, indicating differential induction of splicing variants after development. They also claim that mAB13A4 recognizes two extracellular domains based on Western Blot (+SDS) validation.

While these results may have important implications for the analysis of Prom1 expression, there are several details that are of concern.

It is interesting to note that this occurs in signals that are recognized by antibodies in the post-developmental phase (Fig2). However, this phenomenon is a common one that can be observed in almost all genes with splicing variants. Furthermore, many of the antibodies sold by companies do not reveal the details of the recognition sites, and for these reasons, those conducting research with antibodies are always aware of the effects of splicing variants. Therefore, it is necessary to clarify the biological significance of this phenomenon, which can be approached by examining the following two details.

# We thank the reviewer for carefully reviewing our work. We agree that ascribing a biological function to the alternative isoforms of Prom1 would broaden the appeal of our work. Unfortunately we do not think inclusion of exon 19 is likely to have an effect on the function of Prom1, because of its loss in primates (Figure 1). The significance of our work is that it describes an undocumented behaviour of a widely used reagent. While we agree that the potential of alternative splicing to change the antigenic properties of proteins should be routinely considered by researchers, we find little evidence in the literature that this is the case. In the case of mAB 13A4 this phenomenon remained unnoticed for 25 years after the initial publication, despite the relatively broad use of the antibody. Below are our responses to the specific comments.

1. to clarify whether the difference in signal between mAB 13A4 and ab27699 detected in this study is due to the difference in splicing variants, it is necessary to examine mRNAs from P0 to P110 by PCR or other means. This would also allow us to further discuss the recognition of Exon19 (KFVRVR) by mAB 13A4, which is discussed in Fig. 3 and beyond.

# The switch in splicing of Prom1 in the postnatal retina was shown previously by us. We would like to direct the reviewer to Murphy et al., PLoS genetics 2016, Figure 3 B where we have analyzed the splicing of Prom1 exon 19 in the retina by RT-PCR between postnatal day 0 and day 16. A copy of the figure is included in the PDF version of our response. Inclusion levels of exon 19 switch from mostly skipped at postnatal day 0 to near complete inclusion by postnatal day 12-16. Inclusion levels remain constant after postnatal day 16 based on RNASeq data from the same article and multiple RNASeq datasets published by others. The work by Murphy et al is referenced in this context on page 11 lines 216-219 in the results section.

→Thank you for directing me to the reference. 　As someone who mainly studies retinas, I assume that I do not know the detailed Prom1 behavior during the developmental stages, so I appreciate these changes.

2. it will be important to determine if the proteins detected in mAB 13A4 and ab27699 have any biological significance to maintain the significance of this paper. If the sequence of the splicing variant recognized by mAB 13A4 and ab27699 is revealed, e.g., by method 1, it should be shown experimentally.

# Prom1 has a well established role in stem cells, cancer development and vision. As we review in the introduction mutations in Prom1 are commonly associated with retinal degenerations. We do not think inclusion or skipping of exon 19 has an effect on the function of the Prom1 protein, because the exon is lost in primates (Figure 1B). While the biological significance of Prom1 isoforms containing exon 19 is questionable, our work is significant technically because of the wide use of the mAB 13A4 antibody in cancer and vision research. mAB 13A4 is the most often used reagent for detecting the mouse Prom1 protein with over 300 publications using it. We believe that it is important for investigators to know that the affinity of the antibody is affected by alternative splicing, especially when it is being used for quantitative assessment of Prom1 levels across tissues or developmental time points.

→I understand that the functional significance with or without Ex19 is not important. I support the contributor's assertion.

3. In the description of Fig. 4, it says "Interestingly, under native conditions PROM1 formed higher order complexes corresponding in size to dimers and tetramers. Is this someone else's report? Or is this the content of Fig4C? You should clarify which one it is.

However, if you are referring to Fig. 4C, this data alone does not prove that it is a dimer. To show that it is a dimer or a tetramer, we need Immunoprecipitation, analysis using mutants of the amino acid sequence necessary for Prom1-Prom1 interaction, or analysis of the crystal structure of Prom1. For immunoprecipitation, for example, co-transfection of Flag-Prom1 and HE-Prom1, Immunoprecipitation with Flag and W.B. with HE. The data in Fig. 4C alone does not prove that Prom1 forms homodimers with each other, and the presence of heterodimers such as Prom1-X is also suspected.

# We agree with the reviewer that the data does not prove that these bands are dimers or tetramers. We removed the reference to dimers and tetramers in this sentence (page 14 line 292) and in the Discussion section (page 18, lines 393-394). The two sentences now only point to the existence of higher order complexes without speculating about their composition.

→Confirmed.

5. This paper uses 13A4 and ab27699, what is the difference between them? 13A4 is the hybridoma clone number and ab27699 is the product model number. 13A4 was purchased from ThermoFisher, so it seems correct to use the product model number for 13A4 as well. Even if ab27699 did not have a clone number, it would be misleading not to refer to 13A4 by the product model number. Or is 13A4 using a special purification method?

# The 13A4 is how this antibody was first described and how it is commonly referred to in the literature. The mAB 13A4 name is also how the antibody is recorded in the CiteAB database. In addition, the 13A4 antibody is also available from Sigma-Aldrich under a different part number. Consequently, it will be confusing if we use the vendor part number. In contrast, ab27699 is only available from AbCam and has no other designations.

→I understand that no other designation other than 13A4 is common and that there is no other designation for ab27699.

Overall, I support the publication of this paper.

I apologize for partially misleading you as I have not studied Prom1 as my main focus.

I understood that the content of the paper is of greater benefit to researchers who are dealing with Prom1 than ocular research. I think such a report would be useful in the current situation, where detailed information on antibodies is often not made public by companies and researchers need to find out the details of the antibodies themselves.

I agree that the presence of Ex19 in primates has no biological significance because Prom1 is lost in primates, but I was very interested in the evolutionary significance of the need to lose Ex19 in primates in the first place. They were lost by chance and there may be no significance in losing Ex19, but I thought it would be interesting if the presence or absence of Ex119 was involved in the structure of the primate eye, which has a unique structure compared to other organisms.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

29 Sep 2022

PONE-D-22-16255R1

The mAB 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope

Dear Dr. Stoilov:

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