Mouse monoclonal anti dnmt3b antibody

Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche) for 30 min on ice to extract total protein. Protein concentration was determined by BCA protein assay (ThermoScientific). The protein samples were boiled in 1 × sodium dodecyl sulfate buffer for 5 min. Protein in the same amount was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with TBST (100 mmol/L Tris-HCl pH7.4, 150 mmol/L NaCl, and 0.05% Tween 20) containing 5% nonfat milk for 1 h, followed by overnight incubation with indicated antibody at 4°C. After being washed three times, the membranes were probed by another matching second antibody. Eventually, it was visualized by enhanced chemiluminescence reagents super signal (ThermoScientific). Mouse monoclonal anti-p53 antibody (1:1,000), mouse monoclonal anti-DNMT3b antibody (1:1,500), mouse monoclonal anti-UXT antibody (1:1,000), and mouse monoclonal anti-β-actin antibody (1:2,000) were purchased from Cell Signaling Technology. ImageJ was used for quantitative analysis of gray ribbon.