Fig. 4. Valproate does not induce an ER stress response in HepG2 cells. (A) Protein synthesis rates measured by the incorporation of [³⁵S]methionine/cysteine into proteins during a 15-minute pulse of labeling of HepG2 cells that were pre-exposed to 10 µM A23187 or 0.5 mM valproate for the indicated times. Total protein extracts were resolved by SDS-PAGE and visualized by Coomassie Blue staining (top panel) or autoradiography (middle panel). Total ³⁵S incorporation in each of the tested conditions was determined by scintillation counting and plotted as a percentage of the control count (bottom panel). (B) Immunoblot analysis of protein extracts treated as described in A. Total protein lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were immunostained with antibodies against total eIF2 or the ser-52 phosphorylated form of eIF2. (C) Immunoblot analysis of HepG2 cells cultured for the indicated times in the presence of 0.5 mM valproate or in the presence of 10 µg/ml tunicamycin or 10 µM A23187 for 8 hours. Total protein lysates were resolved and transferred to nitrocellulose membranes. Membranes were immunostained with antibodies against total GADD153/CHOP, ATF4 or ß-actin (loading control). (D) Analysis of ERdj4 mRNA levels in HepG2 cells cultured for the indicated times in presence of 0.5 mM valproate or for 8 hours with 10 µg/ml tunicamycin. Total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes and subjected to blot hybridization with ³²P-radiolabeled cDNA probes encoding ERdj4 and GAPDH (loading control).