Western Blot for Protein Analysis

Western blot experiments was performed as previously described^{24 (link)}. Briefly, protein was extracted with RIPA buffer (Byotime) supplemented with protease inhibitor cocktail (Roche). Total protein concentration was quantified with BCA Protein Assay Kit (Thermo). An equal amount of total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Then block the membrane with 5% non-fat milk, incubated with primary antibodies at 4 °C overnight and followed by secondary antibody for 1 h. The primary antibodies used were as follows: anti-ACSS1 (Invitrogen, PA5-59392), anti-ACSS2 (Cell Signaling Technology, 3568 S), anti-ACSS3 (Invitrogen, PA5-80305), anti-Tubulin (Proteintech, 66031–1), anti-acetyl-histone H4 (Millipore, 06–598), anti-histone H3 (CST, 4499), anti-acetyl-histone H3 (Millipore, 06–599), and anti-histone H4 (CST, 13919).