Fig. 3. SGLT1 protein expression in detergent-resistant membranes (DRM) of proximal tubular cells in primary culture from wild-type animals. (A) Cell membranes from wild-type cells were solubilized in Triton X-100, DRM were purified on a sucrose flotation gradient and an aliquot of each 1 ml gradient fraction (lanes 1-8=5-30% sucrose; lanes 9-12=40% sucrose) was analyzed by western blotting. A rabbit polyclonal anti-SGLT1 antibody and a rabbit polyclonal anti-caveolin antibody were used. (B) SGLT1 protein localization in a Vim^+/+ cell after 4% formaldehyde and ice-cold methanol fixation (left panel) or antibody crosslinking (right panel). A rabbit polyclonal anti-SGLT1 antibody was used, followed by a secondary FITC-conjugated antibody. (C) SGLT1 (left panel) and 5'-nucleotidase (5'-Nu, middle panel) crosslinking in Vim^+/+ cells. An overlay of SGLT1 and 5'-nucleotidase images is shown in the right panel. A rabbit polyclonal anti-SGLT1 antibody and a mouse monoclonal anti-5'-nucleotidase antibody were used, followed by the specific secondary FITC- and TRITC-conjugated antibodies. Finally, cells were fixed in 4% formaldehyde and ice-cold methanol.