Mouse monoclonal anti histone h3

Nuclear extracts or cell lysates (25 μg/lane) were loaded in 1x NuPage loading buffer (Thermo Fisher Scientific, cat # NP0007) onto 12% NuPage® Bis-Tris gels (Thermo Fisher Scientific, cat # NP0336BOX) alongside a Precision Plus Protein dual colour standard marker (Bio-Rad, cat # 1610374). Following standard electrophoresis using 1x MOPS running buffer and transfer, nytran membranes were incubated for 1 hr in blocking solution (TBST, 5% BSA) and probed overnight at 4 °C with mouse monoclonal anti-Histone H3 (abcam, cat # ab10799, 1 in 1000 dilution) or rabbit polyclonal anti-H3K27me3 (Thermo Fisher Scientific, cat # PA5-31817, 1 in 1000 dilution). Detection of EZH2 was performed using rabbit monoclonal anti-EZH2 (Cell Signaling Technology, cat # 5246, 1 in 500 dilution) with normalization against the endogenous control GAPDH (Santa Cruz, cat # sc-47724, 1 in 1000 dilution). Following extensive washes in 1x TBST, membranes were incubated in goat anti-mouse or rabbit IgG –alkaline phosphatase (Sigma Aldrich, 1 in 10000 in blocking solution). Membranes were washed 3 times for 15 min in TBST, followed by signal development in BCIP/NBT liquid substrate (Sigma Aldrich, cat # B1911) with image recording using an Alpha Innotech gel imager (Fluor Chem ® HD2, BioZym), n = 3.

The cells were washed three times with 0.9% NaCl. The cells were lysed in radioimmune precipitation assay buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris pH 8.0, 5.0 mm EDTA, pH 8.0, 0.5 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and phosphatase inhibitor (PhosSTOP; Roche)). Samples were homogenized by sonication (Soniprep 150 MSE) using the following protocol: 14 cycles of 1-s sonication followed by a 1-s interval, amplitude 10. Cell debris was removed by centrifugation. Total protein concentration was determined with a BCA assay (Sigma–Aldrich). Samples were separated by gel electrophoresis and transferred to nitrocellulose membranes by electroblotting. The following primary antibodies were used for immunodetection: mouse monoclonal anti-Viperin (Merck Millipore catalog no. MABF106; 1:500 dilution) and rabbit monoclonal anti-pSMAD2C (Cell Signaling Technologies catalog no. 3108; 1:1000 dilution). As controls, mouse monoclonal anti-Histone H3 (Abcam catalog no. 24834; 1:1000 dilution), mouse monoclonal anti–α-Tubulin (Sigma–Aldrich catalog no. T6074; 1:10,000 dilution), and mouse monoclonal anti-GAPDH (Fitzgerald catalog no. 10R-G109b; 1:5000 dilution) were used. HRP-conjugated polyclonal rabbit anti-mouse (Dako) or HRP-conjugated swine anti-rabbit (Dako) was applied as a secondary antibody, and the bound antibodies were detected by ECL.