Figure 2. The D655V mutation compromises the membrane localization and interaction of OCRL with Rab6. A: HeLaM cells transiently transfected with wild-type (WT) or D655V green fluorescent protein (GFP-OCRL) were fixed, immunolabeled with antibodies raised against the early endosome marker EEA1 (red) and trans-Golgi network protein TGN46 (blue), and examined with immunofluorescence microscopy. Scale bar, 10 µm. B: Glutathione S-transferase tagged (GST-tagged) bait proteins were incubated with extracts from HeLaM cells expressing GFP-tagged WT, D655V, or G664D OCRL, and binding was assessed with western blotting with anti-GFP antibodies. Anti-Pacsin2 and anti-GORAB antibodies were used as positive controls for binding to GST-IPIP27A and GST-Rab6 Q72L, respectively.