Western blot of histones

Histone was extracted from 14-day-old leaves with the following protocols. Nuclei were extracted using Honda buffer (2.5% Ficoll 400, 5% Dextran T40, 0.4 M sucrose, 25 mM Tris–HCl, pH 7.4, 10 mM MgCl₂, 10 mM β-mercaptoethanol, 0.5 mM PMSF, protease inhibitor cocktail) and lysed with histone extraction buffer (10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.25 M HCl, 5 mM 2-mercaptoethanol β-ME, 0.2 mM PMSF). The nuclei lysis was centrifuged and the supernatant was precipitated with 25% TCA. The precipitated pellets were washed by acetone and dissolved by sample lysate (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% NP-40). The extracted histones in 2 × SDS sample buffer were boiled and loaded into SDS-PAGE. Samples were separated by SDS-PAGE, blotted onto PVDF membranes, blocked with 5% milk and subsequently incubated with anti-H3 antibodies (1:5000 dilution; Abcam, ab1791), anti-H3K9me2 (1:2000 dilution; Abcam, ab1220). HRP-linked goat anti-mouse antibodies and HRP-linked goat anti-rabbit antibodies (EASTBIO, BE0101, BE0102) were used as secondary antibodies. ECL prime western blotting detection reagent (GE) was used to induce chemiluminescence.