Fig. 3. Analysis of ER-to-ER exit site (ERES) sorting of VSVG-FP. (A) VSVG-GFP-expressing COS7 cells were incubated for 24 hours at the nonpermissive temperature (40°C) to accumulate VSVG in the ER. Cells were then transferred to ice for 20 minutes and 1 µg/ml nocodazole was added before an additional 20-minute incubation at 40°C. Image analysis was initiated upon transfer of the cells to the permissive temperature (34°C). Images were captured at 20-second intervals. Lower panels, 4x enlarged inserts to show the accumulation in ERESs (see also supplementary material Movie 1). (B) Time-dependent increase in relative PFIVar, calculated as described in the Materials and Methods (black and gray circles) for two representative cells. Data were fitted to an exponential equation (y = ae^kx) (lines). R² values were 0.99 for both data sets, k values were 0.0494 second^–1 and 0.0409 second^–1. (C) Pseudocolor images (upper panel) and surface-plot (lower panel) of a single ERES during cargo sorting (see also corresponding supplementary material Movie 2). (D) Time-dependent increase in average fluorescence intensity of a region of interest within a single ERES, calculated as described in Materials and Methods (black full circles) for a representative ERES. Data were fitted to the sigmoid equation y=864+(1796/(1+e^4.25–0.26x) (lines). R² value is 0.98. Insert is a plot of normalized and averaged data from 10 cells. Error bars indicate s.e.m.