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.