Figure 7.

Quality of nephrin and podocalyxin sialylation influences downstream targets in nls/nls mice. (A) Lectin analysis of nephrin and podocalyxin sialylation. Nephrin and podocalyxin were immunoprecipitated from wt kidney homogenates and analyzed by Western blotting before and after neuraminidase (Neu) treatment (upper panels). α2,6-linked Sia were detected on both proteins with SNA, whereas α2,3-linked Sia was only visible on podocalyxin (MAA staining). PNA detects terminal galactosyl (β-1,3) N-acetylgalactosamine (lower panels). Fetuin served as a positive control for lectin staining. (B) Nephrin phosphorylation and (C) expression of ezrin and phosphorylated ezrin were analyzed in P1.5 kidney homogenates of the indicated genotypes by SDS-PAGE and Western blotting. Cross-reaction with phosphorylated moesin (asterisk) was observed. Actin and GAPDH were used as loading standards. IP, nephrin immunoprecipitate; co, beads without antibody-load served as negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SNA, S. nigra agglutinin; MAA, M. amurensis agglutinin.