Primary anti sirt1 antibody

After HepG2.2.15 cells were treated with 24 μg/mL taraxasterol in DMEM with 2% FBS for 48 h, the cells were harvested and washed using PBS, and RIPA lysis buffer with phenyl-methylsulphonyl fluoride (PMSF) was used to lyse the cell lysates. Protein concentration was tested by BCA assay (Thermo Scientific, Grand Island, NY, USA). Exactly 20 μg of protein was analyzed by standard Western blot procedure [32 (link)]. Cell lysates containing PTBP1 and SIRT1 protein were separated in a 12% polyacrylamide gel with SDS. The proteins in the gels were transferred to a polyvinylidene difluoride membrane (PVDF, Bio-Rad, CA) and detected using primary anti- PTBP1 antibody (Abcam Inc., Cambridge, MA, USA), primary anti- SIRT1 antibody (Abcam Inc., Cambridge, MA, USA), anti-β-actin antibody (Santa Cruz, Dallas, Texas, USA), and secondary antibodies conjugated to horseradish peroxidase, followed by ECL detection. ImageJ was used for the analysis of band intensity (NIH, Bethesda, MD, USA) [4 (link)].

Western blotting analysis was performed from the homogenized frozen biopsy according to Di Filippo et al. [19 (link)]. We used (i) primary anti-SIRT1 antibody (1 : 1000, Abcam, Cambridge, UK), (ii) specific monoclonal antibody directed against FOXO-1 (1 : 500, Millipore, California, USA), (iii) anti-MnSOD primary antibody (1 : 800, Millipore, California, USA), (iv) anti-β-actin monoclonal antibody (1 : 1000, Sigma-Aldrich), and (v) anti-eNOS sc-654 (1 : 500, Santa Cruz Biotech, USA) with an enhanced chemiluminescence detection reagent (ECL), quantified by densitometry using a BioRad ChemiDoc MP Imaging system. The following secondary antibodies were used: goat anti-mouse (1 : 1000, Santa Cruz Biotech, USA) and goat anti-rabbit (1 : 1000, Santa Cruz Biotech, USA). The deacetylase activity of SIRT1 was measured by means of a commercial fluorometric kit (Abcam, Cambridge, UK) and normalized by protein content.