Fig. 7. Characterization of cargo concentration in brefeldin A (BFA) and nocodazole-induced dilated ER exit site (ERES) membranes. (A) Concentration of VSVG-FP in dilated membranes. COS7 cells, transfected with VSVG-FP, were shifted to the permissive temperature after overnight incubation at 39.5°C and 20 minutes at 4°C when 1 µg/ml nocodazole and 5 µg/ml BFA were added. Confocal images were captured at 20-second intervals (see supplementary material Movie 5). (B) Time-dependent increase in relative PFIVar of VSVG-YFP in the absence (average of 12 cells with s.e.m., filled circles) or presence (empty circles) of 5 µg/ml BFA, calculated as described in the Materials and Methods, for the cell in A. Data were fitted to an exponential equation (y = ae^kx) (green and red lines for VSVG-YFP and Sec31-CFP, respectively). R² values were 0.93 and 0.98, and k values were 3.64 and 0.74% per minute, for control and BFA-treated cells, respectively. (C) BFA was added 15 minutes after the shift to the permissive temperature (see supplementary material Movie 6). Scale bars: 20 µm. (D) Three-dimensional reconstruction of a confocal z-section series of a single dilated ERES. (E) Analysis of time-dependent average fluorescence intensity of a single dilated ERES during two cycles of cargo concentration and blink-out. The time zones including an increase in VSVG fluorescence were fitted separately to exponential equations (continuous lines). (F) Pseudocolor images (upper panel) and surface plot (lower panel) of the dilated ERES in E during the time labeled by the blue arrow in E.