H3k9ac

Western blot analysis was performed using as previously described (24 (link)). Briefly, mesenteric arteries were harvested and homogenized in ice-cold RIPA buffer with protease inhibitors. Equivalent amounts of protein (20 μg) were loaded onto 3−8% Tris-Acetate or 4−12% Bis-Tris gels (Invitrogen, USA) for electrophoresis and blotting. Membranes were incubated with the following primary antibodies: p-eNOS (1:2000, BD Biosciences), eNOS (1:2000, BD Biosciences), Nox4 (1:1000, Santa Cruz), SIRT1 (1:500, Cell Signaling Technology), H3K9ac (1:1000, Abcam), H3K14ac (1:1000, Abcam), H3 (1:1000, Abcam) and β-actin (1:2000, Santa Cruz) overnight at 4°C. Membranes were washed before incubating with horseradish peroxidase–conjugated secondary antibodies at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence (ECL) and the Bio-Rad ChemiDOC XRS⁺ imaging system. Band intensity was quantified using ImageJ software.

The antibodies used were as follows: Sirt6 (Abcam, ab62739, 1:1000; CST, 2590, 1:1000), H60b (Santa Cruz Biotechnology, sc-20330, 1:300), β-actin (Sigma, A5376, 1:5000), H3 (Abcam, ab1791, 1:1000), and H3K9Ac (Abcam, ab4441, 1:1000), H3K56Ac (Epigentek, A-4026, 1:1000), H3K14Ac (Abcam, ab52946, 1:1000), MICA/B antibody (R&D, MAB13001, 1:500).

ChIP assays were performed using a commercial kit SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Cell signalling) according to manufacture's protocol. Briefly, MEFs were cross-linked at passage 4 and digested chromatin was immunoprecipitated with 2ug of BAF180 antibody (Bethyl Laboratories, A301-591A), 1 ug of H3K9Ac (Abcam, 4441), 1ug of H3K27Ac (Abcam, 4729), 1 ug of H3K4me1 (Abcam, 8895) and 1 ug of IgG (Cell signaling, 2729). The resulting DNA was used for quantitative real-time PCR using primers located on p21 promoter regions. PM1 (F: 5′-GGGCAGTTTTGACATCCTGT-3′, R: 5′-ACAGGCCTCCTCTTCTGTGA-3′), PM2 (F: 5′-AGTGTGGTCCCAGTCAGGTC-3′, R: 5′-AGACGAGGAAAGCAGTTCCA-3′), −4159/−3958 (F: 5′-GCTTCTGGTTGCCAATGTTT-3′, R: 5′-AAACAGGGCACTTGGTTCAC-3′), Exon1 (F: 5′-CGGTCCCGTGGACAGTGAGCAG-3′, R: 5′-GTCAGGCTGGTCTGCCTCCG-3′).

H3K27ac (39,133, Active Motif Lot # 31814008), H3K27ac (39,685, Active Motif, no. 14517014), H3K18ac (ab1191, Abcam, no. GR3211480–1), H3K27me3 (9756, Cell Signaling), H3K9ac (ab4441, Abcam), H4K16ac (39,167, Active Motif), H3 general (ab1791, Abcam, no. GR177884–2), Spike-In antibody (61,686, Active Motif, Lot# 00419007), p300 (sc-584, Santa Cruz, Lot # F3016), Gapdh (2118, Cell Signaling, Lot # 10), CBP(D6C5) (7389S, Cell Signaling), p53(CM5) (NCL-L-p53-CM5p, Leica Biosystems), PKCs p2056 (ab18192, Abcam), KAP1/TRIM29 pS824 (A300-767A, Bethyl), Acetyl-p53 (Lys379) (2570, Cell Signaling), anti-mouse IgG-HRP (NA93V, GE, no.9773218), anti-rabbit IgG-HRP (170–6515, Bio-Rad, no. 350003248).

Immunofluorescent staining was conducted according
to a previously published study (27 (link)). The experiment was
performed three times. Here, 20 oocytes at MII stage,
were washed in PBS containing 3 mg/ml PVP (PBS/
PVP), fixed for 1 hour in 4% paraformaldehyde in PBS,
and permeabilized by incubation with 0.2% Triton X-100
in PBS for 30 minutes at room temperature. The cells were
blocked in PBS/5% bovine serum albumin (BSA) for 45
minutes and incubated with primary antibody against antiacetyl
H4/K12 (H4K12ac, Abcam, UK) (1:200 dilution)
and H3/K9 (H3K9ac, Abcam, UK) (1:500 dilution) at
4°C overnight. The following day, after washing three
times with PBS/PVA for 5 minutes, the oocytes were
incubated with the secondary antibody conjugated with
FITC for 1 hour at 37°C. Then, DNA was incubated with
3 mg/ml 4, 6-diamidino-2- phenylindole (DAPI) for 20
minutes, and the cells were mounted on glass slides with
etched rings to prevent them from being ruptured by the
cover slip. The slides were imaged using a fluorescent
microscope. Fluorescence intensity was determined by
ImageJ software.