Rabbit anti megalin

For Western blot analysis, rat urine, cell culture supernatants, and cell lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were routinely stained by Ponceau S (Sigma Aldrich Inc.). The primary antibodies were rabbit anti-rat MMP-9 (1:3000), rabbit anti-megalin (1:1000, Abcam), mouse anti-TGF-β1 (1:250, Santa Cruz), and mouse anti β-actin or α–tubulin (1:5000; Sigma Aldrich Inc.). HRP-conjugated secondary antibodies were goat anti rabbit (1:5000), mouse anti-goat (1:5000) and goat anti-mouse (1:5000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Detection was accomplished using enhanced chemiluminescence Western blotting (ECL, GE Healthcare, Piscataway, NJ, USA). For urine and cell culture supernatants, Ponceau red staining was used for loading control. Either β-actin or α-tubulin was used as an internal control for cell lysates. Relative band intensity was measured densitometrically using ImageJ software.