FIG. 2.

CD3 cross-linking activates Raf-1 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. Raf-1 was immunoprecipitated from WCLs and subjected to an in vitro immune complex kinase assay as described in Materials and Methods (top panel). A Raf-1 immunoblot of the Raf-1 immunoprecipitates is shown (second panel). Erk and MEK-1 phosphorylation in WCLs were measured as in Fig. 1 (third and bottom panels). (B) As for panel A, except that 20 μg/of UCHT1 per ml was used and the time of stimulation was varied from 0 to 16 min.