FIG. 3.

CD3 cross-linking activates Ras in P116 T cells. (A) Jurkat and P116 T cells were stimulated by CD3 cross-linking at 37°C for 2 min with purified UCHT1 MAb. The concentration of UCHT1 was varied from 0.2 to 20 μg/ml. Activated (GTP-bound) Ras was purified on the Ras binding domain of Raf-1 (Raf-1–RBD) as described in Materials and Methods. Raf-1–RBD-associated proteins were resolved by electrophoresis on 4 to 12% NuPAGE gradient gels run in morpholinaethanesulfonic acid (MES) buffer, transferred to nitrocellulose, and immunoblotted for Ras (top). Erk phosphorylation in WCLs was measured as in Fig. 1 (bottom). (B) As for panel A, except that OKT3 ascites (1:50) was used and the time of stimulation was varied from 0 to 12 min.