Western Blotting for Proteomic Analysis

For Western blotting, samples were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 6 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, pH 8.0) containing inhibitors of proteases (Roche Diagnostics GmbH, Mannheim, Germany) and centrifuged for 10 min at 14,000× g at 4 °C. In all, 15–50 μg of protein lysates, determined by BCA assay (Invitrogen-ThermoFisher Scientific, Waltham, MA, USA) was denatured and separated by electrophoresis using 8–16% Tris-Glycine Mini Gels (Invitrogen-ThermoFisher Scientific, Waltham, MA, USA) and then electro-blotted onto PVDF membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were blocked with TBS/0.1%-Tween20 (TTBS) containing 5% non-fat dry milk before overnight incubation with the specified antibodies. Peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Jackson ImmunoResearch, Laboratories Inc., Cambridge, UK) were added for 1 h at room temperature in the same buffer as used for the primary antibodies (2.5% non-fat dry milk in TTBS). Reactive bands were detected using Clarity Max^TM Western ECL Substrate (Bio-Rad Laboratories Inc., Hercules, CA, USA), according to the manufacturer’s instructions. Densitometry of Western blot bands was performed with the ImageJ software. Primary antibodies used for Western blotting analysis were as follows: REEP1 (Sigma-Aldrich, St. Louis, MO, USA, #SAB2101976; dilution 1:1000); total DRP1 (BD Transduction Laboratories, Oxford, UK, #611112; dilution 1:500); DRP1 Ser 637 (Byorbyt #orb127984; dilution 1:500). Immunodetection with porin antibody (MitoSciences, Eugene, OR, USA, #MSA05; dilution 1:5000) served as a loading control to normalize the bands intensity.