The GCI tripeptide of RhoB mediates its interaction with Arf6. (A) Schematic of RhoB mutant constructs. (B) HeLa cells were cotransfected with Arf6-HA (WT) and RhoB-GFP (WT), RhoB-GFP (C193S), RhoB-GFP (ΔNCCKVL), or RhoB-GFP (Δ17); lysed; and subjected to immunoprecipitation (IP) using an anti-HA antibody and blotted as shown. (C) HeLa cells were cotransfected with RhoB-GFP (WT), RhoB-GFP (ΔKRYGSQN), RhoB-GFP (ΔGCI), RhoB-GFP (ΔG188), RhoB-GFP (ΔC189), or RhoB-GFP (ΔI190) and then subjected to IP with Arf6-HA (WT) and blotted as shown. (D) HeLa cells were cotransfected with RhoB-GFP (WT), RhoA-GFP (WT), or RhoA-GFP (GCI) and then subjected to IP with Arf6-HA (WT) and blotted as shown. (E) Level of palmitoylated RhoB in the presence of the palmitoylation inhibitor, 2-BP (100 µM, 2 h), or ectopically expressed Arf6-GFP in HeLa cells. CTL, control. (F) IP of endogenous Arf6 in HeLa cells treated with DMSO or 2-BP and blotted as shown. (G) SKBr3 cells were transfected with RhoB mutants as indicated and stimulated with HGF (20 min). Insets show enlargement of the cell leading edge. Representative images of three independent experiments. Scale bar = 10 µm; 5 µm for magnification.