Fig. 5. The DHR-2 domains of the various DOCK180-related proteins have GEF activity toward small GTPases. (A,B) The indicated DHR-2 domains of various DOCK180-related proteins, GST alone or the DPC domain of Vav2 as a positive control were tested in an in vitro GEF assay toward [³H] GDP-loaded Rac1 (A) and Cdc42 (B). Reactions were stopped at 0, 15 and 30 minute time points. Radiolabelled GDP bound to the GTPases was measured by using a filter-binding assay. (C) Schematic representation of the various DOCK2 and DOCK9 constructs that were used to study the in vivo activation of Rac and Cdc42. (D) Various DOCK2 and DOCK9 constructs were expressed in 293-T cells as indicated, and Rac-GTP and Cdc42-GTP were pulled down from cell lysates using the p21-binding domain of PAK fused to GST (PBD pull-down assay). Precipitated Rac 1 and Cdc42 were detected by anti-Rac and anti-Cdc42 immunoblotting, respectively. Transfected proteins were detected by immunoblotting the lysates with anti-Flag or anti-Myc antibodies.