Figure 5

Regulation of eIF2B activity. A. T cells were preincubated with wortmannin (W, 100 nM), rapamycin (R, 100 nM), SB203580 (SB, 10 μM), or PD98059 (PD, 50μM) for 30 min and left untreated (white bar) or the cells were activated with both αCD3 and αCD28 (black bar) for 1 h. Simultaneously cells were activated with PMA (hatched bar) for 1 h. An eIF2B assay was performed as described in Materials and Methods. Bars marked with * are significantly different from the untreated cells (p<0.05, n=5). B. Cell lysates from resting and stimulated cells (1 h αCD3 and αCD28) were analyzed by SDS-PAGE and Western blotting to detect phosphorylated eIF2α. eIF2B was immunoprecipitated from 400 μg of lysate using αeIF2Bε and the amount of protein was analyzed by SDS-PAGE and Western blotting. Similar results were obtained in three experiments C. To test the sensitivity of the eIF2Bε antibody different amounts of T cell extracts (as indicated) were immunoprecipitated with αeIF2Bε and analyzed by SDS-PAGE and Western blotting. Similar results were obtained in two experiments. D. T cells were left untreated (white bar) or the cells were activated with both αCD3 and αCD28 (black bar) or PMA (grey bar) for 1 h. GSK-3α and β were immunoprecipitated together from 400 μg of lysate and a kinase assay was performed as described in Materials and Methods (p<0.05, n=4). E. eIF2Bε was immunoprecipitated from 400 μg of lysate from resting and stimulated cells (1 h αCD3 and αCD28) and the total amount of eIF2Bε and the phosphorylation state of Ser540 were analyzed by SDS-PAGE and Western blotting. Similar results were obtained in two experiments.