FIG. 3.

Effects of amino acids on eIF2α phosphorylation in various cell types. (A to E) The indicated cells were transferred to D-PBS containing or lacking amino acids (AA) for the indicated times (90 min in panel C). Cells were then placed in D-PBS with or without amino acids for 45 min and, where indicated (+R), rapamycin at 100 nM was added, and the incubation was continued for a further 45 min. In some cases, cells were kept in DMEM and treated with thapsigargin (panel D, Tg; 0.5 μM, 1 h) or sodium arsenite (panel E, Ars; 0.5 mM, 30 min). Samples were then analyzed by SDS-PAGE and/or Western blotting for phosphorylated or total eIF2α (A, B, D, and E), for 4E-BP1 phosphorylated at Thr37/46 (C to E), or “total” 4E-BP1 (C).