Hrp conjugated anti mouse or anti rabbit igg secondary antibodies

Cells and tissues were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Sigma-Aldrich). After centrifugation at 13,000 g for 20 min, the supernatant was collected and quantified using a BCA protein assay kit (Thermo Fisher Scientific). Total protein (50 µg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Molsheim, France). After being blocked in 5% non-fat powdered milk, the membranes were probed with primary antibodies at the indicated dilution, followed by the incubation with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Abcam, Cambridge, UK). Protein bands were detected using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and analyzed by GeneTools software (Syngene, Cambridge, UK). The following antibodies were used: anti-Beclin1 (Abcam; dilution 1:1,000), anti-light chain 3 I (anti-LC3 I, Cell Signaling Technology, Danvers, MA, USA; dilution 1:1,000), anti-LC3 II (Cell Signaling Technology; dilution 1:1,000), anti-Bcl-2 (Abcam; dilution 1:1,000), anti-Bax (Abcam; dilution 1:1,000), and anti-VASP (Abcam; dilution 1:3,000). Expression of glyceraldehyde-3-phosphate dehydrogenase was used as a loading control.

After treatment, the cells and mouse kidney tissues were homogenized in RIPA lysis buffer (Cat. No. R0010, Solarbio, Beijing, China) supplemented with protease inhibitors (Cat. No. P6730, Solarbio) and PMSF (Cat. No. R0010, Solarbio). After being centrifuged for 5 min (10,000 g) at 4 °C, the supernatant was collected, and the protein concentrations were quantified using Pierce BCA Protein Assay Kit (Cat. No. 23225, Thermo Fisher Scientific). Protein lysates were mixed with 5× protein loading buffer and boiled for 5 min at 95 °C. Protein samples were fractionated on 4–12% SDS-PAGE gel, transferred to PVDF membranes, and blocked in blocking buffer (5% fresh dry fat-free milk, 0.1% Tween 20 in TBS). Membranes were incubated with primary antibodies (list provided below) overnight at 4 °C. Blots were developed using HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:10000, Abcam) and WesternBright ECL HRP substrate (Advansta San Jose, CA, USA). Densitometry analysis was performed using Quantity One Software (Bio-Rad Laboratories, Hercules, CA), and the protein levels were normalized to β-actin. The details of full length western blots were shown in ‘Supplemental Material’ file.