Analysis of the activation of Rac1 by pull-down assay using PAK2-RBD.

(A) Activation of endogenous Rac1 in Jurkat and K562 cells. Jurkat and K562 cells were transfected with either empty vector or pCXN2-Flag-DOCK2, and drug-resistant cells were lysed and incubated with GST-PAK2-RBD and glutathione-Sepharose beads. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-Rac1 mAb (top and right panels). Expression of DOCK2 proteins was examined by immunoblotting by anti-Flag mAb (right panels). The intensities of precipitated Rac1 were measured and are depicted as a bar graph comparing each with the value of vector control as 1.0. (B-D) The 293T cells were transfected with pCXN2-Flag-Rac1. Expression vectors for the proteins are indicated at the bottom. Cells were lysed and incubated with GST-PAK2-RBD and glutathione-Sepharose beads. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-Flag mAb (top panel and bottom graph). Expression of transfected proteins was examined by immunoblotting by anti-Flag mAb for Vav, DOCK2, and DOCK180, and by anti-CrkL Ab (right panels). The intensities of precipitated Rac1 were measured and are depicted as a bar graph comparing each with the value of vector control as 1.0. Panel B shows activation of Rac1 by CrkL, Vav, DOCK2, DOCK180; panel C, activation of Rac1 by DOCK2 and its truncated mutants; and panel D, suppression of CrkL- and Vav-induced Rac1 activation by the DOCK2 dCS mutant.