Anti podocin

Western blot analysis was performed as previously described (24 (link)). Renal tissues were homogenized with ice-cold lysis buffer and were centrifuged at 10,000 × g for 15 min at 4°C. Equal amounts (100 µg) of protein were loaded and separated by 10% SDS-PAGE. Separated proteins were then transferred onto nitrocellulose paper (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A sample of total protein from the lysate (20 µg) was separated and blotted. The membranes were incubated with the following primary antibodies: Anti-RAGE (1:250, cat no. MAB1179; R&D Systems, Inc., Indianapolis, IN, USA), anti-nephrin (1:1,000, cat no. ab58968; Abcam, Cambridge, UK), anti-podocin (1:1,000, cat no. sc-21009; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin (1:500, cat no. ab8226; Abcam) overnight at 4°C, and then reacted with anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:5,000, cat no. sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 1.5 h. Signal detection was performed via exposure of the blot to enhanced diaminobenzidenecolor reagents (OriGene Technologies, Inc., Rockville, MD, USA) for 5 min. Quantification of the luminosity of each identified protein band was performed using Adobe Photoshop software (AdobePhotoshop 7.0; Adobe Systems, Inc., San Jose, CA, USA).