Western Blot Analysis of Mitochondrial Proteins

Mitochondrial fractions were obtained using the Mitochondria Isolation Kit for Tissue (89801, ThermoFisher Scientific, Waltham, MA). Protein concentrations were measured with the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded into lanes of Criterion TGX (Tris–Glycine eXtended) Stain-Free PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA. The gels were electrophoresed and activated using a ChemiDoc MP Visualization System (Bio-Rad Laboratories, Hercules, CA, USA). The protein was then transferred to a PVDF membrane. The membranes were then imaged using a ChemiDoc MP Visualization System to obtain an assessment of proper transfer and to obtain total protein loads. The membranes were then blocked and probed with primary antibodies overnight at 4 °C. Immunoblots were next processed with secondary antibodies (Cell Signaling) for 1 h at room temperature. Immunoblots were then probed with a Super Signal West Dura kit (Thermo Fisher Scientific) to visualize signal, followed by visualization using a ChemiDoc MP Visualization System (Bio-Rad Laboratories, Hercules, CA, USA). Data were analyzed using Image Lab (Bio-Rad Laboratories, Hercules, CA, USA)^{58 (link)}. Uncropped images of immunoblots are depicted in Supplemental Figs. 1–5.