Figure 3

iAbs induce an Erk-mediated positive feedback loop. A) Purified human T cells were treated with either soluble (sAbs) or immobilized (iAbs) CD3xCD28 mAbs for the indicated time points. Lck expression was detected in cell lysates by anti-Lck immunoblotting. B) Bands corresponding to p56 or p59 Lck were quantified as described in Figure 2. Data represent the ratio of the levels of p56 and p59, and total (p56+p59) Lck shown as arbitrary units ± SEM of 5 independent experiments. C) Purified human T cells were treated with immobilized (iAbs) CD3xCD28 mAbs for the indicated time periods in the presence or absence of MEK Inhibitor I or U0126. Samples were analyzed by Western blotting using the Abs indicated. One representative immunoblot of 4 independent experiments is shown. D) J.CaM1.6 cells were transfected with various constructs carrying different mutations (S42A, S42D, S59A, S59D, S42A/S59A). After transfection, cells were either left unstimulated or stimulated with iAbs for 45 min. Samples were analyzed by Western blotting using the indicated Abs. One representative experiment of five independent experiments is shown.