FIG. 9.
(A) Schematic representation of the proteins used in this study. The polyhistidine tag is depicted as an open box (not in scale). (B) Vav-3 proteins purified from Sf9 cells. (C) (Left panel). Exchange activity of Vav-3 proteins. Equal molar amounts of each Vav-3 protein were incubated with GDP-loaded RhoA in the presence of free [35S]GTP-γS. After 45 min, aliquots of each reaction were taken in duplicate and the exchange determined as indicated in Fig. 3. Similar results were obtained in two independent experiments, each performed in duplicate. (Right panel) Kinetics of GDP-GTP exchange of RhoA either alone (○) or with Vav-3 (Δ1–144) (□) and Vav-3 (Δ1–144+W293L) (▵). (D) Binding of the indicated Vav-3 proteins to RhoA (upper panel) and immunoblot analysis of the same filter with anti-GST antibodies (lower panel). Experiments were conducted as indicated in Fig. 5. The mobility of Vav-3 (DN) and GST-RhoA is indicated by arrows. The blotting antibody is indicated on the right.