Ab115522

The biopsied tissues were initially fixed in 10% buffered formalin, embedded in paraffin and cut into 4 µm sections. Tissue sections were then de-paraffinized and dehydrated in graded ethanol and dimethylbenzene, respectively. Antigen retrieval was performed by boiling sections in EDTA buffer (pH 8.0) for 2.5 min in a pressure cooker. The endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. After rinsing in phosphate-buffered saline (PBS), the sections were incubated for 1 h with polyclonal rabbit anti-DNMT1 antibody (1:200, NB100-264, Novus Biologicals, LLC, Littleton, CO, USA), polyclonal rabbit anti-DNMT3A antibody (1:400, NB100-265, Novus Biologicals, LLC), polyclonal rabbit anti-DNMT3B antibody (1:800, NB100-266, Novus Biologicals, LLC) or polyclonal rabbit anti-DNMT3L antibody (1:100, ab115522, Abcam, Cambridge, UK). After rinse in PBS for several times, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody (UltraSensitive^™ SP kit, Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China) at 37°C for 30 min. Sections were then stained using 3, 3-diaminobenzidine chromogen solution (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and counterstained with hematoxylin (Fuzhou Maixin Biotech Co., Ltd). All slides were observed separately by two pathologists.

The frozen tissue was re-suspended in passive lysis buffer (Promega Corporation, Madison, WI, USA) including freshly added protease inhibitors and phosphatase inhibitors cocktails (Sigma-Aldrich, Darmstadt, Germany). Protein concentrations in the supernatant fractions were determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Protein lysates were separated with 8 or 12% SDS-acrylamide gels, and transferred onto nitrocellulose membranes. After blocking for 60 min in a 5% skim milk solution, membranes were incubated overnight at 4°C with monoclonal rabbit anti-DNMT1 antibody at 1:1,000 (D59A4, Cell Signaling Technology, Inc., Danvers, MA, USA), monoclonal rabbit anti-DNMT3A at 1:1,000 (D23G1, Cell Signaling Technology, Inc.) and polyclonal rabbit anti-DNMT3B at 1:1,000 (NB100-266, Novus Biologicals, LLC), polyclonal rabbit anti-DNMT3L at 1:1,000 (ab115522, Abcam) or monoclonal mouse anti-β-actin at 1:5,000 (A5441, Sigma-Aldrich). Signals from horseradish-peroxidase-conjugated secondary antibodies were visualized by enhanced chemilluminescence solution (GE Healthcare Life Sciences, Chalfont, UK) and processed with an UVP visualizer (UVP, Inc., Upland, CA, USA). Quantification was performed using VisionWorks LS version 7.0 from the UVP visualizer itself. Experiments were repeated in triplicate.