Arf6 directly interacts with RhoB and colocalizes to the same subcellular structures. (A) HeLa cells untreated or treated with 0.5 nM HGF (20 min) were stained for endogenous Arf6 and RhoB. Insets show enlarged region. Arrows indicate multiples vesicles positive for both Arf6 and RhoB. (B) Colocalization between Arf6 (green), RhoB (red), and DAPI (blue) was represented as Pearson’s coefficient and measured for individual HeLa cells (n = 20). (C) Endogenous RhoB coprecipitated with endogenous Arf6 of HeLa cells, which were lysed and subjected to immunoprecipitation (IP) and analyzed by immunoblotting. (D) In vitro GST pull-down assay of purified RhoA or RhoB with purified Arf6, loaded with GDP or GTPγS. (E) HeLa cells were cotransfected with Arf6-HA (WT), Arf6-HA (DA), or Arf6-HA (DN); lysed; and subjected to IP with RhoB-GFP (WT) and Western blotted as shown. (F) HeLa cells were cotransfected with RhoB-HA (WT), RhoB-HA (DA), and RhoB-HA (DN), lysed, and subjected to IP with Arf6-GFP (WT) and Western blotted as shown. (G) HeLa cells transfected with GFP-RhoB (WT), GFP-RhoB (DA), GFP-RhoB (DN), or GFP-GGA3 were lysed and subjected to IP with an anti-GFP antibody; protein complexes were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with fusion protein GST alone or GST-Arf6 and immunoblotted. (H) In vitro binding assay with purified recombinant proteins. GST-RhoB (WT), GST-RhoB (DA), or GST-RhoB (DN) in the presence or absence of His-Arf6, subjected to a GST pull-down and Western blot as shown. (I) Schematic of Arf6 mutant constructs. (J) GST, GST-Arf6, GST-Arf6 28–175, and GST-Arf6 73–175 were transfected into HeLa cells. Protein lysates were then subjected to a GST pull-down and immunoblotted for endogenous RhoB. All quantified data indicate mean values ± SEM from three independent experiments. Scale bar = 10 µm; 5 µm for magnification. P values based on comparisons with control: n.s., nonsignificant.