Fig. 5. Valproate pretreatment does not abrogate an ER stress response. (A) Northern blot analysis of HepG2 cells pretreated for 0, 24 or 48 hours with 0.5 mM valproate and then challenged with 0, 2 or 10 µg/ml tunicamycin (left panel) or 0, 2, 10 µM A23187 (right panel) for 4 hours as indicated. Total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes and hybridized to radiolabeled cDNA probes encoding human GRP78/BiP or GADD153/CHOP. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe. (B) Immunoblot analysis of HepG2 cells treated with valproate, tunicamycin and A23187 as described above. Total protein lysates were resolved and transferred to nitrocellulose membranes. Membranes were immunostained with antibodies against GADD153/CHOP, the ser9-phosphorylated form of eIF2 (eIF2-P) or total eIF2 as indicated.