Protein Extraction and Western Blot Analysis

Western blot was performed according to the method described previously [30 (link)]. Briefly, cytoplasmic and nuclear proteins were extracted from the kidney tissue using the Protein Extraction Kit (KeyGen Biotech Co. Ltd., Nanjing, China), containing protease inhibitor and phosphatase inhibitor cocktails (Sigma Aldrich, Darmstadt, Germany), according to the manufacturer’s protocols. The total protein extracted concentration was considered using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Afterward, 50 µg of the total extracted protein was separated via sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in Tris-buffered saline (TBS), containing 3% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature. After washing with TBS containing 0.1% Tween 20, the membranes were incubated firstly with the primary antibodies (1:300 dilution) for 2 h, and then goat antirabbit HRP-conjugated (as secondary antibody; at a 1:5.000 dilution) at room temperature. The chemiluminescence produced from the luminol reagent was detected with the C-DiGit chemiluminescence scanner (LI-COR, Lincoln, NE, USA), and the band intensity was analyzed using the scanner software.