Lonp1

Antibodies targeting cytochrome c oxidase subunit 1 (COX1), ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), DNAJA3, LONP1, and β-actin were purchased from Abcam (Cambridge, UK); antibody to NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8, mitochondrial (NDUFB8) was purchased from Novex (Carlsbad, CA, USA); and antibodies recognizing succinate dehydrogenase (ubiquinone) iron-sulfur subunit, mitochondrial (SDHB), transcription factor A, mitochondrial (TFAM), HSPD1, and CLPP were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology, and anti-mouse secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Western blotting images were scanned and quantified using an Odyssey imaging system and Image Studio DiGit software (LI-COR Biosciences, Lincoln, NE, USA). Lipid peroxidation was analyzed by measuring malondialdehyde concentrations with a commercial thiobarbituric acid-reactive substances assay kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s specifications.

Mitochondria were isolated from HEK293 cells expressing MTU1 mutants, lysed with ice-cold IP buffer (150 mM NaCl, 25 mM Tris–HCl (pH 7.5), 0.5% Triton X-100) supplemented with protease inhibitor cocktail and centrifuged at 10 000 × g for 10 min. Dynabeads Protein G beads (Invitrogen) were conjugated with primary Myc antibody (MBL) for 1 h at room temperature and were then incubated with the cell supernatant overnight at 4°C. The beads were then washed for 3 times with IP buffer, and proteins were eluted in 1× SDS loading buffer. Eluted proteins were then subjected to western blotting analysis using primary antibodies against MTU1 (Sigma), CLPX (Abcam) and LONP1 (Abcam).