Figure 2

Dependence of eIF2α phosphorylation on PERK in A549 cells treated with cigarette smoke (and thapsigargin. Panel A: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were exposed to air (lanes 1, 3, and 5) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (lanes 2,4, and 6) for 20 minutes. 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 phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. Panel B: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were treated with either 1 uM thapsigargin in DMSO (lanes 2,4, and 6) or DMSO (lanes 1, 3, and 5) for the times specified.