Anti dnmt3b

Total protein extraction from cultured breast cancer cells were performed using RIPA lysis buffer containing 1X protease inhibitor cocktail. After protein concentrations measurement using the BCA method, equal protein (about 20–30 μg) from different samples were separated by 10% SDS‑PAGE and subsequently transferred into the polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Next, the membranes were immersed into 5% non-fat milk diluted in TBST for 1 h at room temperature, followed by incubation with the indicated primary antibodies overnight at 4 °C, including anti‑RSK4 (1:2000 dilution; No.ab76117, Abcam), anti‑SP1 (1:1000 dilution; No. ab227383, Abcam), anti-DNMT1 (1:2000 dilution; No. #5032, Cell Signaling Technology, MA, USA), anti-DNMT3A (1:2000 dilution; No. #2160, Cell Signaling Technology), anti-DNMT3B (1:2000 dilution; No. #67,259, Cell Signaling Technology), anti-Ub (1:1000 dilution; No. #3933, Cell Signaling Technology) and anti-GAPDH (No. ab181602, Abcam). After being washed with Tris Buffered Saline with Tween-20 (TBST) for 3 times, the membranes were probed with the corresponding secondary antibodies at room temperature for 1 h. Band intensity was measured using chemiluminescent reagents (Millipore) and quantified by ImageJ software (verson1.48, National Institutes of Health, MD, USA).

Proteins from HUVECs were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF (Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-μm PVDF membranes (Millipore, Billerica, MA, USA), and incubated overnight at 4 °C with specific antibodies: anti-p16, anti-p21, anti-FIS1, anti-SAHH (Abcam, Cambridge, UK), anti-p53, anti-Drp1, anti-OPA1, anti-MFN1, anti-MFN2, anti-DNMT1, anti-DNMT3A, anti-DNMT3B, and anti-GAPDH (Cell Signaling Technology, Danvers, MA) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA). The dilution ratio was 1:1000 for the primary antibodies and 1:10,000 for the secondary antibodies. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific, Waltham, MA, USA) and GAPDH was used as a control. Images were captured using the FluorChem E system (Protein Simple, Minneapolis, MN, USA) and band densities were quantified using image J software (NIH, Bethesda, MD, USA). The densities of the bands were normalized to that of GAPDH.