Figure 4.

CLT causes activation of PKR and phosphorylation of eIF2α. (A) Exponentially growing NIH 3T3-, PKR-K296-, or eIF2α-51A-transfected cells were labeled with [³²P]orthophosphate (200 μCi/ml) for 3.5 hr. One-half of the cells were challenged with CLT (10 μM) for 30 min, lysed in IP buffer, and RNase–DNase treated. TCA precipitable counts were determined, and equal number of counts were immunoprecipitated with anti-eIF2α antibody and separated by SDS/PAGE, and phosphorylation of eIF2α was quantified by PhosphoroImager. (B) Maternal NIH 3T3 or eIF2α-51A transfectants were lysed and 20 μg of protein was separated by SDS/PAGE and immunoblotted with anti-eIF2α or β-actin specific antibodies.