Visualizing Mitochondrial Dynamics Proteins

Cell extract preparation was performed as described previously (Bienholz et al. 2017 (link)). The shown proteins were visualised using a rabbit antibody against Phospho-Drp1-S637 (Cell Signaling, Danvers, Massachusetts, USA, #4867), Phospho-Drp1-S616 (Cell Signaling, #4494), Drp1 (Cell Signaling, #5391), Opa1 (Cell Signaling, #80471), Fis1 (Thermo Scientific, Waltham, Massachusetts, USA, #PA5-22142), Mfn1 (Merck, Darmstadt, Germany, #ABC41), Caspase-3 (Cell Signaling, #9662), Mff (Cell Signaling, #84580), Phospho-Mff (Cell Signaling, #49281), MiD51 (Proteintech, Rosemont, Illinois, USA, 20164-1-AP), MiD49 (Proteintech, 28718-1-AP) and a mouse antibody against Oma1 (Santa Cruz, Dallas, Texas, USA, sc-515788) and Mfn2 (Abcam, Cambridge, United Kingdom, #ab56889), all in 1:1000 dilution. Anti-rabbit IgG (Sigma-Aldrich, St. Louis, Missouri, USA, #A8275) and anti-mouse IgG (Sigma-Aldrich, #A4416) conjugated with peroxidase was used as secondary antibody in 1:5000 dilution. The chemiluminescence signals (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were recorded using the gel documentation system Fusion Pulse 6 (Vilber Lourmat, Eberhardzell, Germany) and quantified by the software Bio1D (Vilber Lourmat, Eberhardzell, Germany). Gels stained with Coomassie were used as a loading control.