FIG. 4.

(A) Coomassie staining of aliquots of wild-type Vav-3 purified from Sf9 cells. (B) GDP-loaded RhoA (4 pmol) was incubated with [³⁵S]GTP-γS in the presence of 2.0 pmol of either nonphosphorylated Vav-3 (□), nonphosphorylated Vav-3 (Δ1–144) (○), phosphorylated Vav-3 (■), or phosphorylated Vav-3 (Δ1–144) (●). As a negative control, RhoA was also incubated with GST-Lck (◊). Exchange values were determined at the indicated time points as indicated in Fig. 3D. (C) Exchange activity of Vav-3 on Rho/Rac family members by using either 3:1 (left panel) or 1:3 (right panel) molar ratios of GEF-GTPase. The indicated GDP-loaded GTPases were incubated for 45 min at room temperature with [³⁵S]GTP-γS in the presence of GST-Lck (open boxes), nonphosphorylated Vav-3 (gray boxes), or Lck-phosphorylated Vav-3 (closed boxes). Exchange activities were determined as in Fig. 3D. (D) Phosphorylation levels of Vav-3 proteins. Purified wild-type Vav-3 (WT) and Vav-3 (Δ1–144) were separated electrophoretically and subjected to immunoblot analysis with anti-hexahistidine (upper panel) and anti-phosphotyrosine (lower panel) antibodies.