Anti fis1

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.

For detecting oligomeric Drp1, cultured cortical neurons were collected in lysis buffer (100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 50 mM Tris-Cl, protease and phosphatase inhibitor cocktails, pH 7.4). Then, non-reducing SDS-PAGE was performed using samples that were either left untreated or treated with DTT (1 μM). For mitochondrial fractionation, cultured cortical neurons were rinsed with PBS and collected in pre-chilled mitochondria buffer (250 mM sucrose, 10 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES, protease and phosphatase inhibitor cocktails, pH 7.5, 4 °C). Supernatant was obtained by centrifugation at 500 × g for 5 min and 10 000 × g for 15 min at 4 °C. The pellet and supernatant represented the mitochondrial and cytosolic fractions, respectively. Immunoblotting was performed using the following antibodies: anti-Drp1 (1:2000), anti-pDrp1^S616 (1:500), anti-CDK5 (1:1000), anti-p35 (1:1000), anti-β-Actin (Sigma-Aldrich, #A5441; 1:2000), anti-HSP60 (Enzo Life Sciences, Farmingdale, NY, USA, #ADI-SPA-806; 1:1000), anti-GAPDH (Santa Cruz Biotechnology, #6C5; 1:1000), anti-Mfn2 (Abcam, Cambridge, MA, USA, #ab56889; 1:1000), anti-Opa1 (BD Biosciences, #612606; 1:1000), anti-Mff (Sigma-Aldrich, #HPA010968; 1:1000) and anti-Fis1 (Abcam, #ab96764; 1:1000).