Beclin1

Total protein lysates were extracted from mouse renal cortex tissue or cells using RIPA lysis buffer (Solarbio Co., LTD.,Beijing,China). Protein samples were separated by 10-12% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred to polyvinylidene uoride. After being blocked with 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 for 1 h, the membranes were incubated with a primary antibody at 4°C overnight and then with a secondary antibody at room temperature for 1 h. High sig ECL Western blotting substrate development kit was used to develop the imprint. The relative gray intensity of each target protein expression band was quantitatively analyzed by Image J, and GAPDH was set as an internal reference. The ratio of each target protein to GAPDH was the relative expression level of the target protein. All measurements were repeated at least three times. Primary antibodies against SYSTM1/P62, Beclin-1, GSK3β, LC3B, Desmin, and WT-1 were purchased from Servicebio Biotechnology (Wuhan,China), GSK3β (phospho Ser9) was gained from Cell Signaling Technology (Shanghai,China), Nephrin and NPHS2 were purchased from Abcam (Shanghai, China), Sirt1 and phospho Sirt1 (Ser 27) were obtained from Bioss Biotechnology (Beijing, China) , and GAPDH was acquired from Good Here (Hangzhou, China).