Effect of the association of DOCK2 with ELMO1. The association of DOCK2 with ELMO1 is critical for DOCK2-mediated Rac activation and cytoskeletal reorganization. (A) The expression of DOCK2 or DOCK2delN in BEα16-3 (lane 1), N3-5 (lane 2), 17-11 (lane 3), 25-7 (lane 4), and 84-3 (lane 5) was analyzed with the use of anti-DOCK2 antibody. A nonspecific band (NS) is included as a loading control. (B) Cell extracts were prepared from BEα16-3 (lane 1), 25-7 (lane 2), and 84-3 (lane 3); immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2 or DOCK2delN); and analyzed by immunoblotting with anti-ELMO1 antibody (top panel), anti-CrkL antibody (middle panel), or anti-HA antibody (to detect DOCK2 or DOCK2delN) (bottom panel). The expression of ELMO1, CrkL, DOCK2, or DOCK2delN in each cell line is shown in the 3 right lanes of each subpanel. (C) Cell extracts were prepared from BEα16-3 (lane 1) and 17-11 (lane 2), immunoprecipitated with anti-CrkL antibody, and analyzed by immunoblotting with anti-DOCK2 antibody (top panel), anti-C3G antibody (middle panel), or anti-CrkL antibody (bottom panel). The expression of DOCK2, C3G, and CrkL in each cell line is shown in the 2 right lanes. (D) Cell extracts were prepared from 84-3 (lane 1), 17-11 (lane 2), and BEα16-3 (lane 3), and analyzed for Rac or for the GTP-bound, activated Rac by means of glutathione S-transferase (GST)–fusion, Rac-binding domain of p21-activated kinase 1 (PAK1). (E) BEα16-3, 17-11, and 84-3 were analyzed for actin polymerization by means of staining with phalloidin. The differential interference contrast (DIC) images are shown in the left panels. Original magnification, × 600. (F) NS1 (lane 1) and NS1 transfectant expressing ELMO1 (lane 2) were analyzed for Rac (bottom panel) or the GTP-bound, activated Rac (middle panel) as described in panel D. The expression of ELMO1 is shown in the top panel.