Immunoblotting PKC-delta: a cautionary note from the bench.

Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC-delta phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC-delta MAb (generally viewed as an anti-PKC-delta protein antibody) recognizes PKC-delta in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC-delta immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC-delta immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC-delta MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC-delta expression in resting and agonist-activated samples (or in studies of the relationship between PKC-delta phosphorylation and PKC-delta downregulation) because it artifactually displays PKC-delta phosphorylation as a decline in total PKC-delta protein. Other studies have shown that two anti-PKC-delta-pY(311) antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC-delta-Y(311) phosphorylation on the endogenous PKC-delta enzyme, but the Cell Signaling Technology anti-PKC-delta-pY(311) antibody provides a better measure of Y(311) phosphorylation in overexpressed PKC-delta. Collectively, these studies have identified features of anti-PKC-delta antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC-delta may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general.