Fig. 2. SGLT1 and SGLT2 mRNA and SGLT1 protein expression of proximal tubular cells in primary culture from vimentin-null mice (Vim^-/-) and from wild-type littermates (Vim^+/+). (A) Northern blot analysis. Total RNA was extracted from Vim^+/+ and Vim^-/- cultured cells and from whole kidney (K) of Vim^+/+ mice using RNAzol kit. cDNA probes, labeled by a random priming method, were: the rat SGLT1, the rat SGLT2 and the mouse GAPDH. Blots are representative samples from six animals and six separate cultures. (B) Western blot analysis of brush border membranes (BBM). BBM were prepared by MgCl₂ precipitation and differential centrifugation procedures. Proteins were immunoblotted with a rabbit polyclonal anti-SGLT1 antibody and a rabbit polyclonal anti-5'-nucleotidase antibody. Blots are representative samples from five separate cultures. (C) Western blot analysis of biotinylated proteins extracted from BBM. For specific cell surface biotinylation experiments, cells were incubated twice consecutively with NHS-ss-biotin, BBM were prepared and the biotinylated antigens were recovered with streptavidin agarose beads. Then, proteins were immunoblotted with a rabbit polyclonal anti-SGLT1 antibody. Blots are representative samples from two separate cultures. Statistical analysis: no difference was observed between Vim^+/+ and Vim^-/- cells for any of the parameters.