Figure 5.

Vav3 regulates BCR-mediated Akt response through Rac1. (A) Expression levels of wild-type (wt) or GEF mutant Vav3 in Vav3-deficient DT40 cells (left) or of Myc-tagged dominant-negative Rac1 in wild-type DT40 cells (right). Whole-cell lysates were analyzed by Western blotting using anti-Vav3 Abs (left) or anti-Rac1 mAb (right). (B) Rac1 activation in response to BCR cross-linking. Cells (5 × 10⁶) were stimulated with M4 (4 μg/ml), and cell lysates were mixed with beads containing the CRIB of PAK. Elutes of the beads (top) or cell lysates (bottom) were resolved on 12.5% SDS-PAGE and subjected to Western blot analysis with anti-Rac1 mAb. AP, affinity precipitation. (C and D) Effects of mutations of Vav3 and Rac1 on Akt responses. Cells were stimulated with M4 (4 μg/ml) for indicated times. Cell lysates were subsequently subjected to in vitro kinase assay of Akt, as described in the legend to Fig. 4 B. IP, immunoprecipitation.