Anti h3

An aliquot of fixed chromatin after sonication was boiled for 10 min in SDS-PAGE buffer. Proteins were separated on 12% SDS-PAGE gel and transferred to a PVDF membrane (Immobilon-P Transfer membrane, Millipore) by semi-dry blotting in 25 mM Tris–HCl, 192 mM glycine, and 10% methanol. The following antibodies were used: anti-H3K27me3 polyclonal antibody (Diagenode, C15410069), anti-H2AUb (Cell-Signalling Technology, 8240S), and anti-H3 (Agrisera, AS10 710). Horseradish peroxidase-conjugated goat anti-rabbit antibody (Sigma-Aldrich, A0545) was used as secondary antibody at 1/10,000 dilution. Chemiluminescence detection was performed with ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) following the manufacturer’s instructions.

The nucleus fraction of 10-day-old Col seedlings were extracted using Sigma CellLytic PN extraction kit. The soluble cytoplasmic protein was isolated as below: seedlings were ground to fine powder in liquid N2. The powder was further ground in cold grinding buffer (20 mM Tris-HCl (pH 8.8), 150 mM NaCl, 1 mM EDTA, 20% glycerol and cocktail). The sample was spun at 6,000g for 30 min at 4 °C and the resultant supernatant was further spun at 100,000g for 1 h at 4 °C to pellet the total membrane fraction. The resultant supernatant was referred as soluble cytoplasm fraction. Antibodies used include anti-H3 (cat-AS10710, 1:1,000, Agrisera), anti-UGPase (cat-AS142813, 1:1,000, Agrisera), anti-H⁺-ATPase (cat-AS07260, 1:1,000, Agrisera) and anti-PTRE1 (1:1,000).