Anti mitofusin2

Tissues were homogenized in lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, Tris-HCl 20 mM, pH 7.5) containing protease and phosphatase inhibitors. Proteins (15–30 μg/lane) were separated on 10–12% SDS-polyacrilamide gel and transferred to PVDF membranes (GE Healthcare). Blocking (5% non-fat milk, 30 min) and immunoreaction (o/n) were performed in TBST buffer (0.1% Tween, 150mM NaCl, 10mM Tris-HCl, pH 7.5) at room temperature. The primary antibodies were anti-Mitofusin1, anti-Mitofusin2, anti-DRP1 (all Santa Cruz), and anti-β-actin (BD Biosciences). Signals were visualized by horseradish peroxidase-linked secondary antibodies in enzyme-linked chemiluminescence (ECL, Millipore). Relative protein levels were normalized with β-actin levels on the same membrane.

Anti α-tubulin was purchased from Sigma. Anti-LC3, anti-cleaved caspase-3, and anti-VDAC were from Cell Signaling Technology. Anti-p62 was from MBL. Anti-HSP60 was from Enzo Life Sciences. Anti-Drp1 was from Abcam. Anti-Tom20, anti-MID49, anti-mitofusin1, anti-Fis1, and anti-MFF were from Proteintech. Anti-OPA1 was from BD Bioscience. Anti-mitofusin2 was from Santa Cruz Biotechnology. BafilomycinA1 was from LC Laboratories.