Ab52946

Purified histones were electrophoresed in a 17.5% SDS polyacrylamide gel and transferred onto the polyvinylidene difluoride membrane at 15 V for 1 h by the semidry method. The membrane was blocked with TBS-T buffer containing 5% nonfat dried milk at room temperature for 1 h and then washed with TBS-T (twice for 10 min each). Membranes were incubated at 4 °C for 12 h with antibodies (from Millipore unless indicated otherwise) against H3 C-terminal region (07-690), H3K4ac (ABE223), H3K9ac (07-352), H3K14ac (Abcam, ab52946), H3K27ac (07-360), H3K36ac (07-540), H4 C-terminal region (Abcam, ab10158), H4K5ac (07-327), H4K8ac (07-329), H4K12ac (07-595), or H4K16ac (07-328). The membranes were then washed with TBS-T (4 times for 5 min each) and incubated with peroxidase-conjugated anti-mouse IgG (GE Healthcare, NA931) or anti-rabbit IgG (GE Healthcare, NA934) at room temperature for 1 h. The membranes were then washed with TBS-T (4 times for 5 min each) and were detected using enhanced chemiluminescence (Chemi-Lumi One Super: 02230-30).

Following drug treatment, nuclear proteins were isolated from S2 cells and crude protein extracts were made from HEK293 cells. S2 cells were washed once with ice cold PBS and re-suspended in cold nuclear lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl₂ 0.1% IGEPAL). After 5 min incubation on ice the nuclei were pelleted at 700g for 10 min at 4°C. 1× Laemmli buffer was directly added to the pellet and boiled for 10 min at 95°C. HEK293 cells were washed once with ice cold PBS and resuspended in 1x Laemmli buffer and boiled for 10 min at 95°C.
The samples were electrophoresed on 15% SDS-polyacrylamide gel and transferred on to nitrocellulose membrane. Ponceua S stain was used to confirm transfer before blocking and incubating with primary antibodies diluted in TBST–2% BSA: rabbit monoclonal anti-H3K14ac (1:1000, Abcam, ab52946), rabbit polyclonal anti-H3K27ac (1:1000, Abcam, ab4729), rabbit polyclonal anti-Rpd3 (1:2500, gift from Lori Pile) or mouse monoclonal anti-H3 (1:1000, Millipore 05499) overnight. After washing, the membranes were incubated with fluorophore conjugated secondary antibodies IRDye^® 680 RD goat-anti rabbit IgG or IRDye^® 800 CW goat-anti mouse IgG at 1:5000 dilution in TBST–2% BSA. Images were acquired on an Infrared Odyssey System (Li-Cor Biosciences) and bands were quantified using IMAGE studio software.

The cambium from P. trichocarpa stems was harvested for the ChIP assays following our established protocol^{59 (link)}. Anti-FLAG (Sigma, F1804, 5 μg ml⁻¹), anti-H3K9ac (Abcam, ab10812, 5 μg ml⁻¹), anti-H3K14ac (Abcam, ab52946, 5 μg ml⁻¹), anti-H3K27ac (Abcam, ab4729, 5 μg ml⁻¹), anti-PtrVCS2 (Abmart, 6 μg ml⁻¹) or anti-IgG (Abcam, ab205719, 5 μg ml⁻¹) antibodies were used for immunoprecipitation of the fragmented chromatin. The anti-PtrVCS2 monoclonal antibody (against the full-length PtrVCS2 protein) was produced in mice and purified by the IgG affinity chromatography column (Abmart). After immunoprecipitation, the ChIP–DNA was purified and quantified using Qubit Fluorometer. The ChIP–DNA was used for ChIP–qPCR analysis or ChIP-seq library construction. The primers used for ChIP–qPCR are listed in Supplementary Table 6. For ChIP-seq library construction, six libraries (ChIP–DNA and input DNA for three biological replicates) were produced by using the NEBNext Multiplex Oligos for Illumina (NEB, E7335S) and the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) following the manufacturer’s protocol. The ChIP-seq libraries were sequenced using an Illumina NextSeq 500 platform.