Figure 3

The increase in protein synthesis requires signalling via several pathways. T cells were preincubated with wortmannin (W, 100 nM), rapamycin (R, 100 nM), SB203580 (10 μM), SB202190 (10 μM) or PD98059 (PD, 50 μM) for 30 min before activation with both αCD3 and αCD28 for 1 h or the cells were treated with PMA (1 μM) for 1 h. For the last 30 min of activation 10 μCi/ml [³⁵S]-labelled methionine was present. The experiment was performed in duplicate. Protein synthesis in control cells was set at 100%. Methionine incorporation ranged between 600 and 1500 cpm per 50 μg of protein. Incorporation of [³⁵S]-labelled methionine into equal amounts of protein was measured using hot TCA precipitation (p<0.05 for the αCD3/28 and PMA treated samples; all other samples are not significantly different from the control, n=6).