Anti sirt1

The proteins in liver tissues and cultured HepG2 cells were collected, homogenized in radioimmunoprecipitation (RIPA) lysis buffer containing phenylmethylsulfonyl fluoride, and centrifuged at 13,000g for 30 min at 4°C for removal of cellular debris. The protein concentration was determined using a BCA protein assay commercial kit (Thermo, USA). Equal amounts of protein (25 µg) were loaded for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Protein samples were separated by electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membranes. Blots are blocked with 1% bovine serum albumin (BSA, Beyotime Biotechnology, Shanghai, China) or 5% non-fat milk for 1 h and incubated overnight at 4°C with primary antibodies: anti-SIRT1, anti-FOXO1, anti-Acetylated-Lysine and anti-b-actin. All antibodies were from Cell Signaling Technology (Danvers, MA, USA). After washing with the washing buffer, blots were incubated with the corresponding secondary antibody (1:2000, Fcnmacs, Nanjing, China) for 2 h. Photographs were taken, and optical densities of the bands were quantified with the Gel Doc2000 (Bio-Rad, Hercules, CA, USA).