Western Blot Analysis of Mitochondrial Proteins

Western blot analysis was performed as detailed elsewhere.²² (link)^,33 (link) Twenty micrograms of cellular proteins was electrophoresed through 10% Bis-Tris SDS-polyacrylamide gels (Thermo Fisher Scientific) and then transferred to a polyvinylidene difluoride membrane. The primary antibodies used for this experiment were obtained from Abcam (Cambridge, UK), including YARS2 (ab127542), ND1 (ab74257), CO2 (ab110258), TOM20 (ab56783), and total OXPHOS human WB antibody mixture (ab110411), and from Proteintech (Rosemont, IL, USA), including NDUFS1 (12444-1-AP), NDUFA9 (20312-1-AP), NDUFS3 (15066-1-AP), SDHA (14865-1-AP), UQCRC1 (21705-1-AP), COX5A (11448-1-AP), ATP5B (17247-1-AP), and VDAC (55259-1-AP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; AB-M-M 001) was obtained from Hangzhou Goodhere Biotechnology Co., Ltd. (Hangzhou, China), and light chain 3 (LC3; 4108) from Cell Signaling Technology (Danvers, MA, USA). Peroxidase AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG (The Jackson Laboratory, Bar Harbor, ME, USA) were used as a secondary antibody, and protein signals were detected using the ECL Western Blotting Analysis System (MilliporeSigma, Billerica, MA, USA). Quantification of density in each band was performed as detailed previously.²² (link)^,33 (link)