Anti sirt6

Cells were washed with PBS, collected, and lysed on ice for 30 min with modified Radioimmune Precipitation Assay (RIPA) Buffer (Applygen) containing a protease inhibitor cocktail (Amresco). Cell lysates were then centrifuged for 15 min at 12,000 × rpm and 4°C. The supernatant was collected, and the protein concentration was measured using the BCA Protein Assay Reagent (Pierce). Total protein (30-80 μg) was subjected to 10–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and was transferred to nitrocellulose membranes (Millipore). After blocking in 5% non-fat dry milk in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween20), the membranes were incubated with primary antibodies overnight at 4°C. After washing in TBST buffer, membranes were incubated for 1 h in the dark with the appropriate IRDye TM 800-conjugated secondary antibodies (Thermo) in TBST/5% non-fat milk. Signals were detected on an Odyssey Infrared Imager (LI-COR Bioscience) after washing.
The following antibodies were used for the Western blot analysis: anti-SIRT6 (Abcam, CST), anti-Multi Ubiquitin (MBL), anti-FLAG (Sigma), anti-Myc (MBL), anti-p27 (MBL, Santa), anti-GAPDH (Tianjin Sungene Biotech Co., Ltd.), anti-β-TUBULIN (Santa), anti-p21 (MBL), anti-p53 (Santa), anti-p16 (Santa), anti-PTEN (Santa), anti-acetylated lysine (CST).

After treatment, cells were harvested using RIPA buffer with protease/phosphatase inhibitors. The samples were then sonicated for 3 s and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was collected as whole cell extracts and protein samples were loaded on NuPAGE 4–12% Bis-Tris gel (Novex Life technologies) and transferred to PVDF membranes. The membrane was incubated overnight at 4 °C with specific antibodies, such as Anti-SIRT6, anti-γH2AX, anti-acH3K56, anti-acH3K9, and anti-Histone H3 that were obtained from Abcam. Anti-β-actin and anti-p53 were purchased from Sigma-Aldrich and Cell signaling, respectively. The bands were detected using the image analyzer LAS-3000 (Fujifilm) using a western blot detecting kit (Youngin frontier). Band intensities were quantified with Fujifilm Multi Gauge 3.0 Software.

Protein extracts from hepatocytes or liver tissues were made in the lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA and freshly added 1 mM PMSF and an additional protease cocktail tablet from Roche at one tablet/10 mL final buffer volume). Protein extracts were resolved on an SDS-PAGE gel and transferred to nitrocellulose membranes. Immunoblots were blocked in TBST with 5% skimmed milk for 1 hour at RT and incubated overnight at 4°C with the following primary antibodies: anti-NAMPT, anti-Actinin (Proteintech), anti-H3K9Ac, anti-H3K56Ac, anti-histone H3 (Cell Signaling Technology), anti-SIRT1 (Santa Cruz Biotechnology), anti-SIRT6 (Abcam). Following 3 washes in TBST, immunoblots were incubated with HRP-conjugated secondary antibody for 1 hour. After 3 washes in TBST, the immune complexes were detected using the ECL detection reagents (Sigma).