Fluorescein linked secondary antibody

The cortical tissues of the rats’ kidneys and cell lysates were extracted in lysis buffer (KeyGEN Biotech, Nanjing, China). Equal amounts of protein (50 μg) as determined using a BCA assay kit (KeyGEN Biotech, Nanjing, China) were separated on 10 % SDS-polyacrylamide gels and electrophoretically transferred to a nitrocellulose filter membrane (Millipore, Bedford, MA). After incubation with 5 % bovine serum albumin (BSA, dissolved in PBS) for 2 h at room temperature and blocking of the non-specific binding, the membranes were incubated overnight at 4 °C with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at dilution of 1:1000–5000. Then, the fluorescein-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added at a dilution of 1:3000 to the membrane and incubated at room temperature for 2 h. The specific bands were visualized by fluorography using an enhanced chemiluminescence kit (Pierce, Rockford, USA). The relative density was quantified using the Quantity One analysis system (Bio-Rad, California, USA).