Fig. 5. Effects of laminin and GBM substrates on the expression of nephrin in HGEC. Western blotting (A) and confocal microscopy (B) were used to determine nephrin expression. (A) Total protein was isolated from cells cultured either on plastic (lane 1) or dishes coated with 20 µg/cm² of each GBM (lane 2), or laminin (lane 3). 50 µg of total protein were analyzed on SDS-PAGE and immunoblotted with anti-nephrin antibody at a concentration of 2.5 µg/ml in TBS-5% milk. Blots were stripped and reprobed with anti-tubulin antibody (upper panel). Quantification of the protein content of nephrin was performed by densitometric analysis (lower panel). The bars represent the mean±s.d. from three independent experiments after normalizing to the density of ß-tubulin. Results were analyzed using single factor ANOVA and SNK test (*P<0.05 by ANOVA and SNK test; comparison of each different substrate with plastic). (B) For confocal microscopy, cells were cultured on coverslips, either uncoated (control) or coated with substrates, fixed and stained with anti-nephrin antibody at a concentration of 1 µg/ml in PBS and fluorescein-conjugated anti-mouse IgG as a secondary antibody. Panels a-c correspond to X-Y sections of the basal, lateral and apical surface respectively. Scale bar, 15 µm.