Fig. 5. Co-localization and functional interaction of EHD1 with Arf6. HeLa cells were transiently co-transfected with constructs encoding Myc-EHD1 and wild-type Arf6 (A–C), GFP–EHD1 and Arf6-Q67L (D–F), GFP–EHD1 and Arf6-T27N (G–I), GFP–EHD1 and FLAG-EFA6 (J–L), and GFP–EHD1 and FLAG-ACAP1 (M–O). Cells were fixed, permeabilized and incubated with a monoclonal antibody to the Myc epitope and a rabbit polyclonal antibody to Arf6 (A–C). Bound antibodies were revealed by Alexa-488-conjugated antibody to mouse IgG (A and C), and by Cy3-conjugated anti-rabbit IgG (B and C). HeLa cells co-transfected with GFP–EHD1 and Arf6 mutant constructs (D–I) were fixed, permeabilized and incubated with a rabbit polyclonal antibody directed against Arf6 (D–I), followed by Cy3-conjugated anti-rabbit IgG (E, F, H and I). HeLa cells co-transfected with GFP–EHD1 and FLAG-EFA6 (J–L) or FLAG-ACAP1 (M–O) were fixed, permeabilized and incubated with a monoclonal antibody to the FLAG epitope, followed by a Cy3-conjugated anti-mouse IgG antibody. All images were obtained by confocal microscopy. Arrows (A and B) denote tubular structures containing both Arf6 and EHD1. Bar, 10 µm.