Anti mt nd1

Cells were washed with ice-cold PBS twice. Ice-cold RIPA buffer and protease inhibitor cocktail were added next, and cells were scraped from the plate and put into a 1.5-ml Eppendorf tube. Cells were placed on ice for 30 min, then centrifuged at 14,000 rpm for 15 min at 4 °C. Supernatant was collected and frozen at −80 °C until needed. Protein concentration was quantified with the Pierce BCA kit and a NanoDrop2000 (Thermo scientific) to measure. Samples were loaded on a 4–12% bis-tris gel with LDS Sample Buffer (Life technologies) and Sample Reducing Agent (Life technologies). The SDS-PAGE was run with MOPS buffer (Life Technologies), protein was transferred from gel to nitrocellulose membrane, which was then blocked for 1 h at room temperature using 5% BSA. Li-Cor/Odyssey and Image J are used for data collection and analysis. We used the following primary antibodies: anti-MRPL40 (1:500; Novus), anti-VDAC (1:1000; Neuromab), anti-cytochrome b (1:200; Santa Cruz Biotechnology), anti-MT-ND1 (1:500; Abcam), anti-OXPHOS cocktail (1:250; abcam), and β-actin (1:10,000; Cell Signaling Technology). The following Licor secondary antibodies were used all at 1:10,000: IRDye 680LT Goat anti-Mouse, IRDye 680RD Donkey anti-Rabbit, IRDye 800CW Donkey anti-Rabbit, IRDye 800CW Donkey anti-Goat, IRDye 800CW Donkey anti-Mouse.

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).