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Fig. 6. {alpha}V and furin colocalize to the slit diaphragm. (A) Immunogold with the rabbit anti-integrin-{alpha}V antibody and protein-A/10-nm-gold. The gold particles are located over the slit diaphragms (arrowheads) and endothelial fenestrations (arrows). (B) Double immunogold labeling. The {alpha}V (5 nm gold particles) and furin (10 nm gold particles) colocalize to the base of podocytes and over the slit diaphragms (encircled). Bars, 200 nm. (C,D) Total membrane fractions of isolated glomeruli (Memb), treated with DSP (lane 1) or untreated (lane 2), were immunoprecipitated with the anti-{alpha}V-integrin antibody. The resulting material was separated by 10% SDS-PAGE under reducing conditions and analysed by western blotting. (C) The detection of anti-furin antibodies revealed one band of ~98 kDa (arrowhead; lane 1). (D) After stripping the blot shown in C and reprobing it for {alpha}V, one band at ~25 kDa (arrowhead), the molecular mass of the reduced {alpha}V C-terminus, is detected independently of chemical crosslinking (lanes 1,2). This result supports the specificity of the immunoprecipitations. Lane 3, control. Untreated isolated membrane immunoprecipitated with normal rabbit Ig (ctl Ig).