Fig. 2. Involvement of vesicular intermediates in VAMP4-EGFP recycling. NRK cells expressing VAMP4-EGFP (upper panels) or V4nV5-EGFP (lower panels) were incubated with anti-EGFP antibody at 4°C for 1 hour to allow binding to surface fusion proteins. After a brief washing, cells were incubated at 37°C in the absence of antibody for the indicated periods of time to follow the trafficking of fusion protein-bound antibody. EGFP-fusion protein fluoresces green, whereas anti-EGFP antibody was labeled with secondary antibodies conjugated to Cy3 (red). As revealed, recycling of VAMP4-EGFP and V4nV5-EGFP involves peripheral and peri-Golgi vesicular intermediates before being concentrated in the Golgi complex. Bars, 10 µm.