Fig. 2. TCR-mediated PAK1 activation is independent of Slp-76 and Nck. (A) Parental Jurkat cells and the Slp-76-deficient Jurkat subline, J14, were tested for PAK1 kinase activity as in Figure 1 (n = 4). (B) Jurkat T cells were transfected with either 5 µg of pEF-HA-PAK1 (WT), 12 µg pEF-HA-PAK1P13A (P13A) or 5 µg pEFBos (vector) to achieve equal expression levels. Fourteen hours later, the cells were stimulated for 3 min with C305 (+) or a buffer control (–) at 37°C. Anti-Nck immunoprecipitates were analyzed by 7.5% SDS–PAGE and blotted with an anti-HA antibody (top panel) or an anti-Nck antibody (middle panel). Whole cell lysates were blotted with an anti-HA antibody (bottom panel) showing equivalent expression of HA-PAK1. The experiment shown is representative of two independent experiments. (C) As in (B), but anti-HA immunoprecipitates were tested for kinase activity (n = 4). The increased mean fold activation and large range of PAK1 P13A was due to a single experiment out of four in which PAK1 P13A was uncharacteristically activated 3.5-fold above wild-type PAK1.