Anti hdac1

All cell lines were purchased from Cell Bank of Chinese Academy of Sciences. The HEK293T and HT1080 cells were respectively grown in Dulbecco’s Modified Eagle medium (DMEM) or Minimum Essential Medium (MEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS). All cell culture media and supplements were purchased from Hyclone (Hyclone Laboratories). TSA was purchased from Selleck. Anti-Myc (2276S), anti-Flag (14793S), anti-HA (3724S), anti-Trim28 (#5203) were purchased from Cell Signaling Technology. Anti-HDAC1 was purchased from Bethyl. Anti-β-actin (ab3280), anti-H3K9me3 (ab8898), and anti-Trim28 (ab10483) antibodies were obtained from Abcam. Antibody against PFV Gag was kindly provided by Professor Li Zhi, and anti-Tas was produced by immunizing rabbits with Tas and purified according to standard procedures [52 ]. HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies were from Proteintech.

THP-1 cells were treated with variable concentrations of MGCD0103 for 48 h and lysed in Cell Lysis Buffer [20 mmol/L Tris-HCl (pH 8), 0.15 mol/L NaCl, 10% glycerol, and 0.5% NP40] on ice for 2 hours. After centrifugation (12,000 × g for 15 minutes), 500 μg of the supernatant fraction (cell lysate) was incubated with 2 μg rabbit IgG, anti-HDAC1, anti-HDAC3 (Bethyl Labs, Montgomery, TX), anti-HDAC2 (CycLex, Nagano, Japan) or 1000 μg of supernatant fraction was incubated with 2 μg anti-HDAC8 (Santa Cruz Biotechnology, California) overnight at 4°C, followed by incubation with 30 μl of Protein A/G Dynabeads (Life Technologies) for 3 hours at 4°C. The beads were washed three times with ice cold PBS and resuspended in HDAC Assay Buffer [40 mL; 20 mmol/L Tris-HCl (pH 8), 125 mmol/L NaCl, and 1% glycerol] and then HDAC enzymatic activities were measured using the CycLexH HDACs Deacetylase Fluorometric Assay kit (CycLex), or heated at 95°C for 5 min in 30 μl loading buffer for Western blotting.