*2.10. Co-Immunoprecipitation*

To analyze protein acetylation, immunoprecipitation experiments were performed following the recommended protocol of Dynabeads (Invitrogen-Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Proteins containing acetylated lysine (Ac-K) residues were immunoprecipitated from mouse liver mitochondrial extracts using an antibody against acetylated lysine residues (Cell Signaling #9814, Danvers, MA, USA). The immunoprecipitates were separated by SDS-PAGE, blotted onto Amersham Hybond ECL nitrocellulose membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and the Western blots developed using antibody against ANT1 (Abcam #110322, Cambridge, MA, USA) and followed by secondary antibodies. Bands were visualized using the ODYSSEY® CLx (LI-COR Biosciences, Lincoln, NE, USA) infrared scanner.

#### *2.11. Analysis of RCS by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE)*

The RCS in isolated mitochondria were analyzed by BN-PAGE [12,16]. Briefly, NC or ANT1 KD H9c2 mitochondrial protein or rat heart mitochondria treated for 45 min with vehicle (Veh, 0.01% DMSO), 500 nM rotenone (complex I inhibitor), 500 nM antimycin A (complex III inhibitor), or 1 μM FCCP were dissolved in solubilization buffer (50 mM NaCl, 50 mM imidazole-HCl, 2 mM 6-aminohexanoic acid, 1 mM EDTA) supplemented with digitonin, protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA), and 25U benzonase. Native gels were stained with Coomassie brilliant blue G250 and visualized with the ODYSSEY® CLx (LI-COR Biosciences, Lincoln, NE, USA) infrared scanner. The images were analyzed using Image Studio Lite Software. The respirasome levels were calculated as the pixel density of bands containing complex I, III, and IV and normalized to whole lane densities.
