Anti mitofusion2 mfn2

Expression levels of target proteins were measured by western blot as previously described^{44 (link)}. After the indicated treatments, cells were collected and lysed in cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Protein concentrations were determined using a Bradford protein assay kit (Thermo Fisher Scientific). Proteins were electrophoresis by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Anti-Drp1 (1:1000, BD Science), anti-phospho-Drp1(Ser616) (1:1000, Cell Signaling), anti-Opa1 (1:2000, BD Science), anti-Mitofusion2 (Mfn2) (1:1000, Abcam), anti-Fis1 (1:1000, Atlas Antibodies), anti-VDAC (1:2000, Santa Cruz), anti-Bcl-2 (1:1000, Santa Cruz), anti-Bax (1:2000, Santa Cruz), anti-Caspase-3 (1:1000, Santa Cruz), anti-Cleaved Caspase-3 (1:1000, Santa Cruz), and anti-β-actin (1:8000, Sigma) antibodies were used as primary antibodies. The binding sites of primary antibody were visualized with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000, Invitrogen) or anti-mouse IgG antibody (1:5000, Invitrogen), followed by the addition of ECL substrate. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified with NIH ImageJ software (available in the public domain) and normalized with b-actin levels.

Total proteins were extracted with RIPA lysate from the collected HUVECs in each group, and protein concentration was tested by BCA method (Beyotime, China). Protein samples (40 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Roche). PVDF membranes were blocked with 5% skimmed milk powder at room temperature for 2 h and incubated with primary antibody (dilution ratio: 1∶1000) overnight at 4°C and secondary antibody (dilution ratio: 1∶2000) at 37°C for 2 h. The membranes were addressed with ECL solution (Thermo Fisher Scientific), and the results were acquired with a gel imaging system. p-IRAK1 is referenced by IRAK1, p-TAK1 is referenced by TAK1, and GAPDH was applied as the reference for the other proteins. Anti-interleukin-1 receptor-associated kinase 1 (IRAK1), anti-p-IRAK1, anti-TGF-beta-activated kinase 1 (TAK1), anti-p-TAK1, anti-Bax, anti-caspase 3, anti-mitofusion1 (MFN1), anti-mitofusion2 (MFN2) and anti-opticatrophy1 (OPA1) were all purchased from Abcam (USA) [28 (link)].