FIG. 2.
Cellular GADD34 levels following various stress. (A) Time-dependent accumulation of endogenous GADD34 was analyzed in SW480 cells during continued exposure to 1 μM thapsigargin (Tg), 50 μM arsenite (Ars), leucine-deficient DMEM (-Leu), or 5 μM MG132 by immunoblotting with anti-GADD34 antibody as described in Materials and Methods. (B) The schematic shows the experimental design in which cells were subjected to various stress stimuli for 8 h to induce GADD34 (stage I). Then, replacement with complete medium was used to promote the recovery of cells from the stress stimuli (stage II). (C) A549 cells were incubated in media lacking either leucine (Leu) or glucose (Glc) or containing 5 μM MG132 to elicit GADD34 induction. At the times indicated (0, 2, 4, and 8 h) following their transfer into complete media, cells were lysed and GADD34 levels analyzed by immunoblotting as described in Materials and Methods. Immunoblotting with antitubulin antibody was used to assess protein loading.