Fig. 4. The DHR-2 domains of the various DOCK180-related proteins bind to nucleotide-free small GTPases. (A) The in vitro transcribed and translated DHR-2 domain of CED-5 was tested for its ability to interact with nucleotide-free Rac1, Cdc42 or RhoA, as described in Materials and Methods. The bound CED-5 DHR-2 domain was visualized by autoradiography. (B) COS-1 cell lysates were incubated with the indicated DHR-2 fusion proteins or with GST alone as described in Materials and Methods, and the precipitated Rac1 was visualized by anti-Rac immunoblotting. (C) The DHR-2 domains of DOCK7 and DOCK9 were in vitro transcribed and translated as in (A) and tested for their ability to interact with nucleotide-free Rac1, Cdc42 and RhoA. The DHR-2 domains were visualized by autoradiography.