Phosphorylation of eIF2α in leucine-deprived ES cell is dependent upon GCN2. Gcn2+/+ (A) and Gcn2−/− (B) ES cells were grown in leucine-replete (lane C) or leucine-deficient (−Leu) media as described in Materials and Methods. Cells were harvested at 1, 3, 6, 9, and 12 h and analyzed by immunoblotting with antisera directed against either eIF2α or eIF2α[P]. The ratio of eIF2α[P] to eIF2α is indicated at the bottom of each panel. For comparison, Gcn2+/+ and Gcn2−/− ES cells were also treated for 1 or 3 h with 1 μM thapsigargin (Tg) to induce eIF2α phosphorylation via the PERK-dependent unfolded protein response.