Figure 4.
Enhanced recovery of client protein synthesis predis-poses wild-type cells to accumulation of a high-molecular-weight ER protein complex during ER stress. (A) Autoradiograph and Coomassie stain of gel of radiolabeled microsomal proteins isolated by flotation through sucrose from [35S]methionine pulse-labeled wild-type and CHOP-/- MEFs treated with 400 nM thapsigargin. (B) Autoradiograph (top) and Grp94, BiP, and PDI immunoblots (bottom) of proteins from the wild-type 6-h time point of A following velocity gradient centrifugation through a 10%-40% glycerol gradient. (C) BiP immunoblot from untreated and tunicamycin-treated (2μg/mL, 8 h) wild-type cells. A total of 5% of the input, post-nuclear supernatant (PNS), or the pellet obtained from that post-nuclear supernatant treated with the indicated concentration of SDS and subjected to glycerol gradient centrifugation were loaded in each lane. (D) Immunoblot of BiP from fractions taken through the glycerol gradient from a 0.8% SDS-treated sample as in C (note that BiP migrating through the cushion as a large complex forms a distinct peak that is separated by a clear zone from the low-molecular-weight material). (E) Immunoblot of pelleted BiP (as in D) and 5% of the input post-nuclear supernatant (PNS) BiP from untreated and tunicamycin treated wild-type, CHOP-/- and GADD34 mutant cells.