Sirt6

Protein extracts from tissues were made in tissue lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, and freshly added 1 mM PMSF and an additional protease cocktail tablet from Roche at one tablet/10ml final buffer volume). Protein extracts were resolved on an SDS-PAGE gel and transferred to nitrocellulose membrane (Santa Cruz Biotechnology). The membrane was incubated with the following antibodies: SIRT6 (Sigma-Aldrich), Actinin (Santa Cruz Biotechnology), AKT, p-AKT(S473), and AdipoQ (Cell Signaling Technology). Detection of proteins was carried out by incubations with the HRP-conjugated secondary antibodies, followed by the ECL detection reagents (Thermo Fisher Scientific).

Embryos or cells were lysed by sonication in lysis buffer (Cell Signaling Technology, #9803) with a Protease inhibitor cocktail (Sigma) (Wang et al. 2015e (link), Yang et al. 2013 (link), Gu et al. 2015a , Wang et al. 2015a (link), Wang et al. 2015d (link)). Equal amounts of protein from embryos or cells were resolved by SDS-PAGE gel electrophoresis and transferred onto Immunobilon-P membranes (Millipore, Billerica, MA). 2 μg Precision Plus Protein Standards (Bio-Rad, Hercules, CA) were loaded into one lane of the gel. Membranes were incubated in 5% nonfat milk for 45 minutes and then incubated for 18 hours at 4°C with the following primary antibodies at dilutions of 1:1000 to 1:2000 in 5% nonfat milk: SIRT2, SIRT6 from Sigma-Aldrich (St. Louis, MO), acetylated H3K56, H4K16, H4K9, and H3K27 from Cell Signal Technology (Danvers, MA). Membranes were then exposed to goat anti-rabbit or anti-mouse secondary antibodies. To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and probed with a mouse antibody against β-actin (Abcam, Cambridge, MA). Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Scientific, Rockford, IL). All experiments were repeated three times.

Protein extracts from cells were made in tissue lysis buffer (50mM Hepes, pH 7.5, 150mM NaCl, 10% glycerol, 1% Triton X-100, 1.5mM MgCl2, 1mM EGTA) and an additional protease cocktail tablet from Roche at one tablet/10ml final buffer volume. Protein extracts were resolved on an SDS-PAGE gel and transferred to nitrocellulose membrane (Santa Cruz Biotechnology). The membrane was incubated with the following antibodies: Sirt6 (Sigma-Aldrich), Actinin (Santa Cruz Biotechnology), Ac-H3K9, Cleaved Caspase 3 (Cell Signaling Technology). Detection of proteins was carried out by incubation with HRP-conjugated secondary antibodies, followed by ECL detection reagents (Thermo Fisher Scientific).