Brag2 and Dock180 deficiency decreases GTPase activity. (A) Western blot analysis of total expression of Rac in each cell line after d 3 in DM. Rac migrates as a 21-kD protein and GAPDH migrates as 35-kD protein. (B) Western blot indicating the level of activated Rac, as detected in lysates at d 3 in DM using a pull-down assay with GST-fused PAK protein-binding domain. Lysates from CntSi cell line were treated with GTPγS and GDP for positive and negative controls, respectively. (C) The histogram represents the levels of GTP-Rac as determined by three independent experiments. All samples were normalized to total protein and Brag2Si, Dock180Si, Cnt2Si, and DKD values are also normalized to the Rac-GTP levels observed in CntSi. (D) Western blot analysis of total expression of ARF6 after d 3 in DM. ARF6 migrates as a 20-kD protein. (E) Western blot indicating the level of activated ARF6, as detected in lysates at d 3 in DM using a pull-down assay with GST-fused GGA3 protein-binding domain, which specifically binds ARF6-GTP. (F) The histogram represents the levels of ARF6-GTP as determined by three independent experiments. All samples were normalized to total protein and Brag2Si, Dock180Si, Cnt2Si, and DKD values are also normalized to the ARF6-GTP levels observed in CntSi at d 3 in DM.