Anti cbx4

Protein was extracted from the fresh ACP surgical specimens by lysing cells with protease inhibitor cocktail (Roche, USA). After measuring the protein content with a Bradford assay, 20 μg of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and incubated overnight with the following antibodies: anti-HAT, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8 (1:1000, Abcam, USA), anti-CBX4, anti-Runx2, anti-histone (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:1000, Proteintech, USA). The specimens were then incubated with secondary antibodies, IRDye800-conjugated anti-rabbit IgG and IRDye680-conjugated anti-mouse IgG (1:15,000, LiCor, USA), for 1 h at room temperature to label the primary antibody. An Odyssey Infrared Image System (LiCor, USA) was used to analyze signal intensities. The densitometry results were first normalized to the density obtained for β-actin or histone and then compared with that of the control to obtain relative fold changes.

Proteins were separated by SDS–PAGE and blotted onto nitrocellulose membranes (0.45 µm, Thermo Fisher Scientific) using a Bio-Rad PROTEAN mini-gel and blotting system. Antibodies were diluted in TBST + 5% milk (25 mM Tris (pH 7.5), 137 mM NaCl, 2.7 mM KCl, 0.2% Tween-20, 5% non-fat dry milk). The following primary antibodies were used for immunoblots: anti-V5 tag (eBioscience, TCM5 14-6796-82, 1:1,000), anti-INO80 (Abcam, ab118787, 1:2,000), anti-INO80B (Santa Cruz (E-3), sc-390009, 1:1,000), anti-ACTR5 (GeneTex, GTX80453, 1:1,000), anti-TBRG1 (Santa Cruz (D-9), sc-515620, 1:1,000), anti-H3K4me3 (Millipore, 17-614, 1:2,000), anti-H4 (Abcam, ab31830, 1 µg ml⁻¹), anti-H4ac (pan-acetyl) (Active Motif, 39967, 1:1,000), anti-CBX4 (Cell Signaling Technology, E6L7X 30559, 1:1,000), anti-CBX8 (Santa Cruz (C-3), sc-374332, 1:1,000), anti-H2B (Abcam, ab1790, 1:1,000), anti-H2A.Z (Abcam, ab4174, 1:1,000). Immunoblot images were acquired by CCD camera using the Bio-Rad ChemiDoc Touch Imaging System running Image Lab Touch Software (v.2.3.0.07).