Figure 1
Activation of T cells with αCD3 and αCD28. A. Primary T lymphocytes were activated with both αCD3 and αCD28 for 4, 8, 16 or 24 h and for the last 45 min 10 μCi/ml of [35S]-labelled methionine was present. The experiment was performed in duplicate. Incorporation of [35S]-labelled methionine into equal amounts of protein was measured. Protein synthesis in control cells was set at 100%. Methionine incorporation ranged between 1000 and 2000 cpm per 50 μg of protein. (control cells at t = 0 h and t = 24 h are not significantly different, n = 3). B. T cells were activated by αCD3 and αCD28 or with PMA. Cells were activated for 30 min, harvested and 50–80 μg of lysate was analyzed by SDS-PAGE and Western blotting. Antibodies that recognize the phosphorylated form of either ERK (pp42) or p38 MAPK (pp38) were used. Even loading of the gel was verified using anti-ERK2 (p42). Similar results were obtained in three sets of experiments. C. PKB activity was measured as described in Materials and Methods (p<0.05, n=4).