Fig. 3. Inhibition of internalization of VAMP4-EGFP by treatments blocking clathrin-mediated endocytosis. (A) After a hypotonic shock, NRK cells expressing VAMP4-EGFP were incubated with anti-EGFP in the absence (a-f) or presence (g-l) of K⁺ for 30 (a-c and g-i) or 60 minutes (d-f and j-l). EGFP-fusion protein fluoresces in green, while anti-EGFP antibody was labeled with secondary antibodies conjugated to Cy3 (red). The merged images are also shown (c,f,i and l). (B) After hypertonic treatment, NRK cells expressing VAMP4-EGFP were incubated with anti-EGFP in the presence (a-f) or absence (g-l) of 0.45 M sucrose for 30 (a-c and g-i) or 60 minutes (d-f and j-l). EGFP-fusion protein fluoresces in green and anti-EGFP antibody was labeled with secondary antibodies conjugated to Cy3 (red). The merged images are also shown (c,f,i,l). Bars, 10 µm.