Ab81215

Muscle biopsies and cybrid cells were lysed in cell lysis buffer (Beyotime, China). 30‐50 mg protein were run on a 8‐12% sodium dodecyl sulfate‐polyacrylamide gel (SDS–PAGE) at 100 V, 2‐3 h. Proteins were transferred to PVDF (Immobilon‐P PVDF‐Membrane, Millipore, Burlington, MA, USA) for 1‐3 h at 100 V, 4°C. After blocking with 5% dried skimmed milk, the membrane was incubated with various primary antibodies. Primary antibodies were as follows: total OXPHOS antibody cocktail (ab110413, Abcam), anti‐CO4 (CST 4805s, Cell Signaling Technology), anti‐GAPDH (10494‐1‐AP, Proteintech), anti‐ND5 antibody(ab92624, Abcam), anti‐ATP6 antibody (ab192423, Abcam), anti‐ND1 antibody (ab181848, Abcam), Anti‐CYB antibody (ab81215, Abcam), Anti‐VDAC1/Porin antibody (ab158995, Abcam), Anti‐β actin antibody (TA‐09, Zhongshan Jinqiao).

B16 cells were transfected with Myg1 SiRNA using Dharmafect (SMARTpool:ON-TARGETplus Myg1 siRNA, L-055338-01-0005). After 24 h, the cells were trypsinized and lysed using NP40 lysis buffer supplemented with protease inhibitor cocktail. For detection of mitochondrial protein, 40 μg of cell lysate was resolved on 10% SDS-PAGE gel. Antibody was prepared in 2% skimmed milk solution with following dilutions MT-CO1(Abcam, ab90668, 1:500), MT-ND5(Abcam, ab92624, 1:500), MT-ATP6 (Abcam, ab192423, 1:100), MT-CYB(Abcam, ab81215, 1:100), Myg1 (Abcam, ab122493, 1:2000), Tubulin (Abcam, Ab21058, 1:5000). It was detected using HRP conjugated anti rabbit secondary antibody (1:10 000) and detected through Enhanced Chemiluminescence using ChemiDoc (Syngene).