Tri methyl histone h3 lys27 c36b11

HCT116 cells (1.5 × 10⁶ cells) were seeded into 10-cm cell culture dishes and incubated for 24 h. Then cells were treated with DZNep (5 µM) for 1, 3, 6, 24, and 48 h. Untreated HCT116 cells were served as control. After the indicated incubation periods, cells were harvested, washed in PBS, and proteins were extracted by lysis with Urea Lysis Buffer. Protein concentration was measured using the Bio-Rad DC™ Protein Assay (BioRad). Equal amounts of lysates were applied to SDS-PAGE and proteins were then transferred to a nitrocellulose membrane (Amersham Protran Premium 0.2 NC, GE Healthcare Life Sciences) prior to probing with antibodies. Antibodies were applied as follows: anti-EZH2 (1:10,000, EZH2 (D2C9) XP, monoclonal rabbit, Cell Signaling), anti-H3K27me3 (1:10,000, Tri-Methyl-Histone H3 (Lys27) (C36B11, monoclonal rabbit, Cell Signaling), anti-GAPDH-HRP as loading control (1:50,000, clone 6C5, monoclonal mouse, Abnova), and secondary HRP-conjugated antibody (1:10000, Goat anti-Rabbit IgG (H + L, Thermo Scientific). Signal of protein bands was detected using chemiluminescent HRP substrate (Immobilon™ Western Chemiluminescent HRP substrate, Millipore) according to the manufacturer’s instructions and the GeneGnome detection system (Syngene).

Whole-cell pellets were lysed in RIPA buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Complete; Roche) and phosphate inhibitors (10 mM NaF final concentration, 1 mM Na₃VO₄ final concentration, 25 mM β-Glycerophosphate final concentration, 1 mM PMSF, and 1 mM Na₄P₂O₇ final concentration), and 20 mM DTT. Protein concentrations were measured on a Nanodrop 2000c spectrophotometer (ThermoFisher) using Protein Assay Dye reagent (Bio-rad). Protein was loaded in equal amounts onto 4–12% Bis-Tris gels (NuPage-Novex, Invitrogen) and transferred onto nitrocellulose membranes (0.2 μm; Whatman). Membranes were blocked in 5% BSA in phosphate-buffered saline (PBS) with 0.1% Tween-20 (PBST) for 1 h, incubated with primary antibodies in PBST 1% BSA overnight at 4 °C, and incubated with secondary antibodies coupled to HRP for 45 min in PBST 1% BSA at room temperature. Amersham ECL detection reagent was used for antibody detection (GE Healthcare). Imaging of the membranes was done on a Bio-Rad ChemiDoc XRS + . The following antibodies were used for western blot analyses: BAP1 D7W70 (Cell Signalling, 13271S), p-ATM (Ser1981) (BioLegend, 651201), Tri-Methyl-Histone H3 (Lys27) C36B11 (Cell Signalling, 9733S), anti-Tubulin (Sigma, T9026).