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Fig. 4. FRAP analysis of VSVG diffusion and reverse ER exit site-to-ER flux. (A) Cells expressing VSVG-GFP were treated with nocodazole as described previously. 25 minutes after shift to the permissive temperature, most of the cell was photobleached, apart from the area circled in yellow. Images were captured at 20-second intervals for an additional 20 minutes. Lower panel, inverted and magnified images of the boxed areas in the upper panel (see also corresponding supplementary material Movie 3). Scale bar: 10 µm. (B) Quantitative analysis of the average and total (insert) fluorescence intensities of the bleached (open circles) and unbleached (closed circles) regions of interest. Lines in the insert were generated by simulation using SAAM II software. The simple model used is illustrated below the graph. The rate constant obtained from the fit is 4% per minute. Insert was fitted to a linear equation (continuous line) indicating an increase in total fluorescence intensity of 0.23% per minute. (C) FRAP analysis of VSVG-GFP in the ER of nocodazole-treated COS7 cells. The rectangle photobleaching, image acquisition and extraction of the apparent diffusion coefficient are described in the Materials and Methods. Insert is a full-log-scale graph to show the quality of fit at all time points.
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