Polyvinylidene di uoride membranes

Mitochondria were isolated from cells with a mitochondria extraction kit (Solarbio, Beijing, China) according to the manufacturer's instructions. BN-PAGE was performed with the NativePAGE TM system (Invitrogen, CA, USA). In brief, mitochondria were solubilized with digitonin (digitonin/protein ratio of 4 g g -1 ), incubated on ice for 30 min, and centrifuged at 20000 × g for 30 min. The solubilized protein was collected from the supernatant, and the protein concentration was determined by the BCA (Solarbio, Beijing, China) method. Protein samples were mixed with Coomassie blue G-250 (Invitrogen, CA, USA) to obtain a dye/detergent mass ratio of 1/4 and were then loaded into a 4-16% Bis-Tris gel. After electrophoresis, proteins were transferred to 0.45-μm polyvinylidene di uoride membranes (Invitrogen, CA, USA) and were then sequentially probed with antibodies against complex I (NDUFB8), complex III (RISP), complex IV (COX IV), complex II (SDHA) and complex V (ATPB). The NDUFB8, RISP and COX IV immunoblot bands with high molecular weights were used to identify the complex I-, complex III-, or complex IV-containing respiratory supercomplexes (RSCs). Signal intensities were normalized to their corresponding ATPB signals.

Mitochondria were isolated from cells with a mitochondria extraction kit (Solarbio, Beijing, China) according to the manufacturer's instructions. BN-PAGE was performed with the NativePAGE TM system (Invitrogen, CA, USA). In brief, mitochondria were solubilized with digitonin (digitonin/protein ratio of 4 g g -1 ), incubated on ice for 30 min, and centrifuged at 20000 × g for 30 min. The solubilized protein was collected from the supernatant, and the protein concentration was determined by the BCA (Solarbio, Beijing, China) method. Protein samples were mixed with Coomassie blue G-250 (Invitrogen, CA, USA) to obtain a dye/detergent mass ratio of 1/4 and were then loaded into a 4-16% Bis-Tris gel. After electrophoresis, proteins were transferred to 0.45-μm polyvinylidene di uoride membranes (Invitrogen, CA, USA) and were then sequentially probed with antibodies against complex I (NDUFB8), complex III (RISP), complex IV (COX IV), complex II (SDHA) and complex V (ATPB). The NDUFB8, RISP and COX IV immunoblot bands with high molecular weights were used to identify the complex I-, complex III-, or complex IV-containing respiratory supercomplexes (RSCs). Signal intensities were normalized to their corresponding ATPB signals.