Gt1189

Western blotting was performed as previously described (21 (link)). Mouse kidney and cultured cells were lysed in RIPA lysis buffer. Anti-GAPDH (1:2000 dilution, CW0100M, CWBio, China), anti-cleaved caspase 3 (1:1000 dilution, ab32503, Abcam, Cambridge, UK), anti-Bcl2 (1:1000 dilution, cst2870, Cell Signaling Technology, USA), anti-SIRT1 (1:1000 dilution, GT1189, Genetex, China),anti-KIM-1(0.25ug/ml, AF1817, RD systems, Canada), anti-PGC-1a(1:1000, # 2178, cell signaling technology, USA) and anti-Bax (1:1000 dilution, ab193349, Abcam Cambridge, UK) antibodies were used in this study. Experiments were repeated three times.

Briefly, for the immunohistochemical staining of SIRT1, tissue sections were boiled in EDTA antigen retrieval buffer. After cooling, sections were incubated to block endogenous peroxidase activity with 3% hydrogen peroxide solution at room temperature for 15 min. Sections were then incubated overnight at 4°C in a humid chamber with an antibody against SIRT1 (1:200 dilution, GT1189,Genetex, China). Then, tissue sections were incubated with a biotinylated secondary antibody for 1 h. Finally, sections were counterstained with hematoxylin.