Rabbit anti lc3b

Mesangial cells were cultured in 12-well plates with coverslips. After receiving various treatments for 48 h, cells were fixed with 4% paraformaldehyde at room temperature for 20 min and washed 3 times with PBS for 15 min. Then cells were permeabilized with 0.1% Triton X-100 for 30 min at 4°C. After being blocked with goat serum for 2 h, cells were incubated with primary antibodies overnight at 4°C, namely rabbit anti-LC3B (GeneTex, United States), rabbit anti-PINK1 (Abcam, United Kingdom), mouse anti-Parkin (Santa Cruz Biotechnology, United States). After that, cells were washed 3 times for 15 min and incubated with fluorescent secondary antibody (Zhuangzhi, China) for 2 h in the darkroom at room temperature. Eventually, the nuclei of cells were stained with DAPI and images were captured by a fluorescence microscope (Olympus, Japan).

Total protein of renal tissues and glomerular mesangial cells was extracted by RIPA lysis buffer supplemented with protease inhibitors (Solarbio, China). Protein concentrations were determined using the BCA Protein Kit (Solarbio, China). All samples were boiled at 95°C for 5 min. Equal amounts of proteins were separated in 8–12% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, United States). After blocked with 5% non-fat milk for 2 h, the membranes were incubated with various primary antibodies overnight at 4°C, namely rabbit anti-LC3B (1:1,500, GeneTex, United States), rabbit anti-P62 (1:1,000, Proteintech, China), rabbit anti-PINK1 (1:1,000, Abcam, United Kingdom), mouse anti-Parkin (1:200, Santa Cruz Biotechnology, United States), rabbit anti-Drp-1 (1:1,000, Cell Signaling Technology, United States), rabbit anti-Mfn-2 (1:1,000, Cell Signaling Technology, United States). And then the membranes were washed 3 times with Tris-buffered saline containing Tween-20 for around 15 min and subsequently incubated with corresponding secondary antibodies for 2 h. After washing 3 times with TBST for around 15 min, the bands were detected by using ECL reagents (Abbkine, United States) and their density was quantified by using ImageJ software.