Ab181848

Western blotting analysis was performed as detailed previously.^{32 (link)} Antibodies used in this study include anti-YARS2 (ab228957, at a dilution of 1:1000), anti-MT-ND1 (ab181848, at a dilution of 1:2000), anti-MT-ND5 (ab230509, at a dilution of 1:1000), anti-MT-CO2 (ab79393, at a dilution of 1:5000), and anti-TOM20 (ab186735, at a dilution of 1:2000) from Abcam, anti-MT-ND6 (PA5-43532, at a dilution of 1:1000) and anti-MT-ND4 (PA5-97298, at a dilution of 1:1000) from ThermoFisher, anti- MT-CYTB (55090-1-AP, at a dilution of 1:1000) from Proteintech, anti-β-actin (AF5003, at a dilution of 1:2000) from Beyotime Institute of Biotechnology, and HRP-conjugated anti-rabbit secondary antibody (A0208, at a dilution of 1:1000) from Beyotime Institute of Biotechnology. Immunoreactive proteins were visualized using a chemiluminescent immunodetection system (ChemiDoc XRS). Image J was employed to analyze the grayscale values obtained by western blotting.

Total protein was extracted from cybrids using the EzRIPA Lysis Kit (ATTO, Tokyo, Japan). SDS-PAGE was performed using NuPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific) and MOPS Running Buffer (Thermo Fisher Scientific). Then, the proteins were transferred to PVDF membrane. After blocking with a PVDF Blocking Reagent (TOYOBO, Osaka, Japan), membranes were incubated with the following primary antibodies: anti-NDUFA9 (ab14713, Abcam, Cambridge, UK), anti-MT-ND1 (ab181848, Abcam), anti-SDHA (#11998, Cell Signaling Technology), anti-COX IV (#4850, Cell Signaling Technology), anti-MT-COI (ab14705, Abcam), anti-Hsp60 (#12165, Cell Signaling Technology), anti-mtHsp70 (#3593, Cell Signaling Technology), anti-Tid1 (#4775, Cell Signaling Technology), anti-Lonp1 (NBP1-81734, Novus Biologicals, Littleton, CO), and anti-α–Tubulin (T-5168, Sigma-Aldrich). All primary antibodies described above were used at 1:1000 dilution. As the secondary antibodies, horse radish peroxidase-linked anti-mouse IgG (at 1:3000, #7076, Cell Signaling Technology) or anti-rabbit IgG (at 1:3000, #7074, Cell Signaling Technology) was used. The membranes were then incubated with ECL substrate (Thermo Fisher Scientific). Signal detection and quantification were performed using the ImageQuant LAS4000 (GE Healthcare) and MultiGauge software (FUJIFILM, Tokyo, Japan).