Anti h4k5ac

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.).

For in vitro histone acetyltransferase activity assay, vectors of HAG704, ACLA2, ACLB-3×FLAG, and H4-6×His were transformed into tobacco (Nicotiana benthamiana) leaf epidermal cells. 48 h after the transfection, the transfected tobacco leaves were collected and ground into powder by liquid nitrogen. Then the proteins were extracted with lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and purified with Ni-NTA Magnetic Agarose beads (QIAGEN, 36113). The extracted proteins were boiled at 90 °C for 10 min and then analyzed using Western blotting. The primary antibodies used were anti-HAG704 (1:1000, in house), anti-H3 (1:1000, Abcam, ab1791), anti-H3K9ac (1:1000, Millipore, 07–352), anti-H3Kac (1:1000, Millipore, 17-615), anti-H4 (1:1000, Abcam, ab177840), anti-H4K12ac (1:1000, PTM-Biolab, PTM-121), anti-H4K5ac (1:1000, Millipore, 07-327), anti-H4K16ac (1:1000, Millipore, 07-329), anti-ACLA2 (1:1000, homemade), anti-FLAG (1:1000, Sigma, F3165). The secondary antibodies used were peroxidase-conjugated goat anti-rabbit antibody (1:10000, Abbkine, A21020), and peroxidase-conjugated goat anti-mouse antibody (1:10000, Abbkine, A21010). The original raw blotting images are shown as a Source Data file.