Kinetics of ATF6 and PERK with persistent ER stress. (A) HEK293 cells, expressing FLAG-tagged ATF6α, were treated with tg (500 nM) for the indicated times, and ATF6f was detected by immunoblotting. GAPDH levels served as a protein loading control. (B) HEK293 cells were treated with tg, and phospho-PERK, phospho-eIF2α, and ATF-4 levels were determined by immunoblotting. Total eIF2α levels served as a protein loading control. In the bottom panels, cells were treated with drug for the indicated times and were pulse-labeled, and radioisotope incorporation was measured via phosphoimaging. 35S-Met/Cys, 35S-labeled methionine/cysteine. (C) Cells were treated with agents (tm, red bars; tg, blue bars) for the indicated hours, and normalized BiP (top panel) and Chop (bottom panel) mRNA levels were measured by quantitative PCR and shown relative to levels in untreated cells. Error bars represent SDs from five independent experiments.