Fig. 4. GADD34 is required for eIF2α dephosphorylation and translational recovery during ER stress. (A) Upper panel, autoradiogram of an SDS–PAGE gel showing 35S-methionine/cysteine incorporation into newly synthesized proteins in untreated (UT) and thapsigargin (Tg)-treated wild-type and GADD34ΔC/ΔC fibroblasts. Arrowheads to the right of the autoradiogram indicate the ER stress-inducible chaperones GRP78 and GRP94. Lower panels, immunoblots of P-eIF2α and total eIF2α, and immunoblots of PERK immunoprecipitates from the same cells. (B) Graphic presentation of 35S-methionine/cysteine incorporation into newly synthesized proteins (left graph), and fold induction of phosphorylated eIF2α (right graph) in untreated and thapsigargin-treated wild-type and GADD34ΔC/ΔC fibroblasts. The level of 35S incorporation in untreated cells is set at 100% whereas the signal of phosphorylated eIF2α from untreated wild-type cells is set as 1. Shown are mean ± SEM of experiments performed in duplicate and reproduced twice.