Representative Western blot analysis of nephrin in non–cross-linked mAb 5-1-6 and RVG-1 immunoprecipitations. (a) A Triton X-100 glomerular extract was incubated with either mAb 5-1-6 or RVG-1 followed by Protein G-agarose. The washed pellets were solubilized in reducing sample buffer and resolved by 7.5% SDS-PAGE. After electroblotting to nitrocellulose, the membrane was incubated with rabbit antinephrin (1:1,000) followed by HRP-conjugated goat anti-rabbit IgG (1:2,000). Reactive proteins were detected using a chemiluminescent technique. A specific antinephrin reactive protein of 185 kDa is present in the mAb 5-1-6 precipitate lane and is absent from the control lane. (b) A Triton X-100 glomerular extract was incubated with mAb 5-1-6 followed by Protein G-agarose. The washed pellet was solubilized in reducing sample buffer and resolved by 5% SDS-PAGE together with equal samples (10 μL/lane) from the glomerular extract before and after the mAb 5-1-6 IP. After electroblotting to nitrocellulose, the membrane was incubated with rabbit anti-nephrin (1:1,000) or normal rabbit Ig (1:1,000) followed by HRP-conjugated goat anti-rabbit IgG (1:5000). Reactive proteins were detected by chemiluminescence (antinephrin: 1- to 2-second exposure; normal rabbit Ig: 30-second exposure). In addition to the 185-kDa band in the mAb 5-1-6 precipitate lane, there is a double band in the glomerular extract lanes with partial depletion of nephrin from the postprecipitation lane (densitometry of the upper band pre- versus postprecipitation: 174 ± 20.9 versus 137 ± 24.0; n = 5, mean ± SEM, P < 0.02, paired t test).