Anti sirt

Liver samples (about 0.06 g) were digested with 300 μL of lysis buffer (mixed with a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland)). Samples containing 30 μg of proteins were subjected to SDS–PAGE gels and transferred to a PVDF membrane (0.45 µm or 0.22 µm, for 100 min at 100 V). The membranes were blocked with 5% bovine serum albumin (BSA; BioShop, Burlington, ON, Canada) for 1 h and then probed with primary antibodies at 4 °C overnight, including anti-PPAR-γ, anti-SIRT, and anti-p-NF-κB, which were purchased from Cell Signal Technology (Beverley, MA, USA). Anti-horseradish peroxidase (HRP)-conjugated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Proteintech (Rosemont, IL, USA). Subsequently, the membranes were incubated with the corresponding goat anti-rabbit antibody IgG (Abcam, Cambridge, UK) for 1 h. The chemiluminescence was imaged using the eBlot Touch Imager^TM (eBlot Photoelectric Technology, Shanghai, China) after being reacted with electrochemiluminescence (ECL; Thermo Fisher Scientific, Waltham, MA, USA). Densitometric estimations were quantified using the ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA). All of the raw data obtained using Western blotting are presented in Supplementary Figure S1.

Whole cell protein extracts were prepared according to Laemmli [56 (link)]. The primary antibodies used were: anti-ATM (1:500), anti-phospho-ATM Ser1981 (1:500) (Abcam, Cambridge, UK); anti-GAPDH (1:50000) (Millipore, Darmstadt, Germany); anti-p53 (1:500) (Santa Cruz Biotechnology, Santa Cruz, USA); anti-p21 (1:500) (Sigma-Aldrich, St. Louis, USA); anti-phospho-p53 Ser15 (1:250), anti-sirt 1 (1:250), anti-phospho-sirt 1 Ser47 (1:250), anti-sirt 3 (1:500), anti-sirt 5 (1:500), anti-sirt 6 (1:1000), anti-sirt 7 (1:250) anti-AMPKα (1:500), anti-phospho-AMPKα Thr172 (1:1000), anti-phospho-ACC Ser79 (1:1000) (Cell Signaling Technology, Denvers, USA). The respective proteins were detected after incubation with the horseradish peroxidase-conjugated secondary antibodies (1:2000) (Dako, Glostrup, Denmark), using an ECL system (Thermo Scientific, Rockford, USA, according to the manufacturer's instructions.