Effect of CS on proteolytic cleavage of ATF6. Panel A: A549 cells were transfected with a plasmid expressing ATF6 (lanes 2 and 4, indicated by +), or a control plasmid (lanes 1 and 3), then treated with 2 mM DTT for 1 h (lanes 3 and 4) or left untreated (lanes 1 and 2). Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Panel B: All lanes show lysates from A549 cells transfected with a plasmid expressing ATF6. Cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Gel bands were quantified as described in the Methods section. CS treatment resulted in a 23% and 26% decrease in ATF6 90 kDa protein at 2 and 4 h respectively (lane 5 compared to lane 6, lane 7 compared to lane 8).