Quantitative measurement of events in the mammalian unfolded protein response.

When the homeostasis of endoplasmic reticulum (ER) is disturbed by the accumulation of unfolded or misfolded proteins, a series of signaling responses collectively called the unfolded protein response (UPR) is triggered. UPR transducers IRE1, PERK, ATF6, and UPR-responsive genes such as GRP78/BiP, ERAD genes such as EDEM, and synthesis of the protein N-linked glycosylation donor lipid-linked oligosaccharides (LLOs) are mobilized. This chapter provides methods used in our laboratory to quantitatively measure the accumulation of mRNAs encoding BiP and EDEM, splicing of XBP1, cleavage of ATF6, inhibition of protein synthesis by PERK, and extension of LLOs under control and stress conditions.