FIG. 4.

Interaction between Vav and Rho family GTPases. Jurkat-TAg cells were transfected with the indicated plasmids (5 μg each). After 24 h, the cells either were left unstimulated or were stimulated with cross-linked OKT3. (A) The active, GTP-loaded forms of Rac1 or Cdc42 in cell lysates were captured by incubation with a GST-PBD fusion protein, followed by immobilization on glutathione-coupled Sepharose 4B beads. Washed precipitates were analyzed by immunoblotting with the indicated antibodies (top two panels). Total cellular extracts from the same groups were immunoblotted with anti-Vav, anti-Rac1, or anti-Cdc42 antibodies (bottom three panels). The results shown are representative of five separate experiments. (B) Cell lysates were immunoprecipitated (IP) with anti-Rac1 or anti-Cdc42 antibodies or with control IgG and immunoblotted with an anti-Vav MAb. Total cellular extracts were immunoblotted as described for panel A. The results shown are representative of three separate experiments. The positions of molecular weight standards (in thousands) are shown.