Anti mfn1

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.