Fig. 7. Vimentin protein expression in detergent-resistant membranes (DRM) of proximal tubular cells in primary culture from wild-type animals. Cell membranes were solubilized in Triton X-100, then DRM were purified on a sucrose flotation gradient. (A) An aliquot of each 1 ml gradient fraction (lanes 1-8=5-30% sucrose; lanes 9-12=40% sucrose) was collected and analyzed by western blotting using a mouse monoclonal anti-vimentin antibody. (B) Fractions 4-7 of DRM were pooled, washed, resuspended in TNE buffer containing 1% Triton X-100 and a second sucrose gradient was performed as described above, followed by western blot analysis. A mouse monoclonal antivimentin antibody and a rabbit polyclonal anti-caveolin antibody were used. (C) The pooled 4-7 DRM fractions were immunoprecipitated with a rabbit polyclonal anti-caveolin antibody (lines 1 and 2) or non-immune rabbit serum (line 3). The immunoprecipitates were analyzed by western blotting using either a rabbit polyclonal anti-caveolin antibody (left and right) or a mouse monoclonal anti-vimentin antibody (middle). (D) Effect of methyl-ß-cyclodextrin (MCD) on DRM vimentin expression. Cells were treated or not with MCD (10 mM at 37°C for 2 hours), then DRM were prepared as described above, followed by western blotting using a mouse monoclonal anti-vimentin antibody. All blots are representative samples from three separate cultures.