Rabbit anti β actin

Total protein extracted by RIPA buffer was used to detect the expression levels of all molecules. General use protease and phosphatase inhibitor cocktails (50×, Beyotime Biotechnology, Shanghai, China) were added to the lysis buffer for protein extraction. The proteins (10 μg) were separated with 10–12% SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% nonfatty milk for 1 hour at room temperature and then incubated at 4°C overnight with the following primary antibodies: rabbit anti-Rho-kinase (1:1000; Abcam, UK), rabbit anti–vimentin (1:1000; CST), rabbit anti-p-NF-κB-P65 (1:1000; CST), rabbit anti-p-IκBα (1:1000; CST), rabbit anti-nephrin (1:1000; Abcam, UK), rabbit anti-podocin (1:1000; Abcam, UK), and rabbit anti-β-actin (1:1000; BOSTER, China). Next, the membranes were washed with TBST buffer and incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 hour at room temperature. Protein bands were detected using an ECL system and a Bio-Rad electrophoresis image analyser (Bio-Rad, Hercules, CA, USA). β-actin was used as internal total protein control.

Total protein was extracted from cells using RIPA buffer (Biosharp, China), and then the protein concentration was quantified using a bicinchoninic (BCA) protein assay kit (Beyotime, China). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk for 1 h and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit anti-LC3 (Cell Signaling Technology, 12741), rabbit anti-Beclin1 (Cell Signaling Technology, 3495), rabbit anti-ATG7 (Cell Signaling Technology, 8558), rabbit anti-P662 (Cell Signaling Technology, 16177), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, 9664), rabbit anti-caspase 3 (Cell Signaling Technology, 14220), rabbit anti-Bax (BOSTER, A00183), rabbit anti-Bcl-2 (BOSTER, A00040-2), rabbit anti-β-actin (BOSTER, BA2305). Afterward, the membranes were incubated with secondary antibody at room temperature for 1 h. An ECL kit (Beyotime Biotechnology, Shanghai, China) was used to detect the bands of the western blots.