Hdac3

Suberoyl anilide hydroxamic acid (SAHA, a pan-HDAC inhibitor) and 3-Deazaneplanocin A (DZNep, a polycomb EZH2 subunit inhibitor) were purchased from Millipore, Temecula, CA. The DNA methylation inhibitor 5-AZA-Cytidine (AZA) was from Sigma, St. Louis, MO. Cells were treated with indicated concentration and incubated for 72 hours before harvesting. Agarose A/G plus is from Santa Cruz. The following antibodies were used as primary antibodies: SOX4 (Santa Cruz; 1:1000); EZH2 (BD; 1:4000); HDAC3 (Millipore; 1:4000); total histone H3 (Abcam; 1:5000); Tri-methylated histone H3 H3K27me3 (Millipore; 1:3000); SUZ12 (Santa Cruz; 1:1,000); EZH1 (Santa Cruz; 1:1,000); β-tubulin (Sigma-Aldrich; 1:5,000).

Equal amounts of protein lysates from HUVECs was harvested and lysed by SDS-PAGE and transferred to PVDF membrane (Merck Millipore, IPVH00010), then subjected to western blotting analysis: Membranes were blocked with 5% (v/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) and incubated with primary antibodies overnight at 4℃. The proteins were visualized using an Image Quant LAS 4000 (GE Healthcare) system, and the secondary antibodies are: goat anti-rabbit HRP (Bio-Rad, 1706515), goat anti-mouse HRP (Bio-Rad, 1706516).
For nuclear Nrf2 accumulation assays, HUVECs were harvested and lysed to obtain cytoplasmic and nuclear lysates using the Keygen Nuclear-Cytosol Protein Extraction Kit from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China).
The primary antibodies used to probe the membranes included Nrf2 (Santa Cruze Biotechnology, sc365949), Bax (Abcam, ab32503) and Bcl-2 (Abcam, ab59348) and GAPDH (CST, 5174), Lamin B (CST, 12,586), Keap1 (CST, 4678), c-Caspase-3 (CST, 9661), HDAC3 (CST, 85057), eNOS (CST, 32027), acetylated-lysine (CST, 9441), 3-NT (Millipore, 05–233), Nox4 (Origene, TA349083).

After removal of the culture medium, the cells were washed with cold 1X PBS and were lysed using a lysis buffer supplemented with protease and phosphatase inhibitors: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 40 mg/mL phenylmethylsulfonyl fluoride (PMSF), 20 g/mL aprotinin, 20 mg/ml leupeptin, 2 mg/mL antipain, 10 mM p-nitrophenyl phosphate, 10 mg/mL pepstatin A and 20 nM okadaic acid. Cells were then centrifuged at 13000 rpm for 15 minutes at 4°C, and protein content of supernatant was used to determine the protein concentration by colorimetric assay (Biorad, Italy). Cell extracts were diluted 1:1 in sample buffer 2X Laemmli (0.217 M Tris-HCl pH 8.0, 52.17% SDS, 17.4% glycerol, 0.026% bromo-phenol blue, 8.7% beta-mercapto-ethanol), and then boiled for 3 minutes. Equal amounts of protein (50 μg) were run and separated by SDS-PAGE gel (acrylamide gel). Primary antibodies used were HDAC1 (Santa Cruz), HDAC2 (Alexis), HDAC3 (Sigma), CIITA (Abcam), all diluted 1:500; 100mg/ml anti-ERK1 antibody (Santa Cruz Biotechnology) was used for normalization.