Fig. 7. Arrest of VAMP4-EGFP recycling in peri-Golgi REs when vacuolar ATPase is inhibited by BFLA1 or conA. (A) NRK (j-o) or VAMP4-EGFP-expressing NRK (a-i) cells were pre-incubated with DMEM alone (a-c), in the presence of 50 nM BFLA1 (d-f,j-l), or in the presence of 100 nM conA (g-i,m-o) for 30 minutes. Cells were then incubated with anti-EGFP and Tf-AF647 (a-i) or Tf-FITC alone (j-o) for 30 minutes in the absence or presence of the drug as indicated. Fluorescent dye-conjugated Tf is shown in green, internalized anti-EGFP (b,e,h) or endogenous GM130 (k,n) is shown in red. Panels l₁ and o₁ are enlarged images of the indicated portion of the merged images, l and o, respectively. (B) VAMP4-EGFP-expressing NRK cells were pre-incubated with either 50 nM BFLA1 (a-c) or 100 nM conA (d-f) for 30 minutes. Cells were then incubated with anti-EGFP antibody and Tf-AF647 in the continuous presence of either drug for an additional 30 minutes, washed and subjected to a further 30-minute incubation in fresh DMEM, without the drug at 37°C. Tf-AF647 signal is green and anti-EGFP signal is red (see above). Bars, 10 µm.