Fig. 5. VAMP4-EGFP recycling was arrested at the peri-Golgi recycling endosomes (REs) at 18°C. Cells expressing VAMP4-EGFP were incubated at 37°C (a-c) or at 18°C (d-i) with either anti-EGFP alone (g-i) or both anti-EGFP and Alexa Fluor 647-conjugated transferrin (Tf-AF647) (a-f) for 1 hour. Anti-EGFP is red (b,e,h) while Tf-AF647 (a,d) or endogenous LBPA (revealed by antibody labeling) (g) is green. The merged images are also shown (c,f,i). Panel f₁ shows the enlarged image of a part of panel f. As a control, NRK cells were incubated at 18°C with FITC-conjugated transferrin (Tf-FITC) for 1 hour (j-l). Cells were then analyzed to view the internalized transferrin (j) and endogenous GM130 as revealed by antibody labeling (k). The merged image is shown in panel l. Panel l₁ shows the enlarged image of a part of panel l, indicating that the peri-Golgi RE are segregated from the Golgi complex. Panels m-o show another control experiment where cells expressing VAMP4-EGFP were incubated at 18°C with Tf-AF647 (green) and anti-EGFP (red) for 1 hour. Cells were then incubated in DMEM at 37°C for an additional 30 minutes before being fixed for viewing. Bar, 10 µm.