Anti h4k12ac

The ground fine powder of each time point was mixed with extraction buffer containing 100 mM Tris, pH 8.0, 1.5 M β-mercaptoethanol, 4% SDS, 15% glycerol, 2 mM EDTA, 0.005% bromophenol blue, 2 μM/mL Trichostatin A (TSA) and vortexed for 30 s. The mixed solution was boiled for 5 min at 99 °C and vortexed 30 s. The supernatants were transferred into a new collection tube after centrifugation at 15,000 g for 10 min at RT. Proteins were loaded into 12% sodium dodecylsulfate-polyacryamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to a polyvinylidene fluoride (PVDF) membrane, and probed using anti-H3 (ab1791) and anti-H3K18ac (ab1191) from Abcam, anti-H3K23ac (#07–355), anti-H3AC (#06–599), anti-H4K5ac (#07–327), anti-H4K8ac (#07–328), anti-H4K12ac (#07–595) and anti-H4K16ac (#07–329) from Millipore at a dilution of 1:2000 (v/v) in 5% skim-milk TBST (10 mM Tris-HCl, pH 75, 150 mM NaCl, and 0.1% Tween 20) for 1 h at RT after incubation with blocking buffer for 1 h at RT. The membranes were washed three times with TBST for 5 min each. Next, membranes were incubated with secondary antibody (AS014, ABclonal, Inc) in 5% skim-milk-TBST for 1 h at RT. Signals in membranes were detected using the BLT Gel View 6000 Plus machine (Biolight Biotec Co., Ltd.).

Rice spikelets were used to extract histone‐enriched proteins as described previously (Lu et al., 2018 (link)). Histones were analysed by immunoblotting using anti‐H3 (PTM‐1001), anti‐H3K9ac (PTM‐112), anti‐H3K27ac (PTM‐116), anti‐H3K36ac (PTM‐117), anti‐H3K56ac (PTM‐118), anti‐H4 (PTM‐1004), anti‐H4K5ac (PTM‐119), anti‐H4K8ac (PTM‐120), anti‐H4K16ac (PTM‐122) antibodies (PTM BioLabs, Hangzhou, China), and anti‐H4K12ac (Millipore, MA, USA, 04‐119). ImageJ software was used for quantification of the band intensities from the immunoblots.