Chemiluminescence imager

All solutions, tubes, and centrifuges were maintained at 4°C. RIPA buffer (Cell signaling, #9806) with 1% SDS and protease inhibitor (Complete Mini, Roche) was used to extract total protein lysates from tissues or cells according to the manufacturer’s instructions. Protein concentrations were measured using BCA protein assay (Pierce, #23225). Lysates were heated (95°C) for 10 minutes in Laemmli sample buffer (Bio-Rad). Proteins were then separated by electrophoresis in acrylamide gels (8–15%) and transferred using a Bio-Rad western system to nitrocellulose (Bio-Rad) membranes. Transferred blots were blocked for 1h in 5% non-fat milk in Tris-buffered saline. Membranes were incubated with specific primary antibodies at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated anti-mouse antibody (1:5,000), or anti-rabbit antibody (1:5,000) at 25°C for 1 h. Resulting immunoblots were visualized using ECL Western Blotting Substrate (Pierce) in LI-COR chemiluminescence imager (LI-COR), according to the manufacturers’ instructions.

Western blot assays were performed to detect protein expression in kidneys or cells. Briefly, whole cell, nuclei or cytosol lysates (10 ~ 50 μg of protein) were heated (95 °C) for 5 minutes in laemmli sample buffer (Biorad). Proteins were then separated by polyacrylamide gel electrophoresis in acrylamide gels (8~15%) and transferred using a Bio-Rad western system to polyvinylidene difluoride (PVDF; Biorad) membranes, which were immediately placed in 5% non-fat milk in Tris-buffered saline (TBS, 50 mM Tris, pH 7.6, 150 mM NaCl)-Tween (0.1% Tween20) buffer for blocking (1 h at 25 °C). Membranes were then washed 3 times in TBS-Tween buffer for 10 minutes, followed by incubation with specific primary antibodies (dilution 1:500 to 1:2000) at 4 °C overnight. Membranes were then washed 3 times for 10 min in TBS-Tween buffer, and incubated with a horseradish peroxidase-conjugated anti-mouse antibody (1:10,000), or anti-rabbit antibody (1:10,000) at 25°C for 1 h. Resulting immunoblots were visualized using ECL Western Blotting Substrate (Pierce) and a LI-COR chemiluminescence imager (LI-COR), according to the manufacturers’ instructions.