Blue native polyacrylamide gel electrophoresis
Blue native polyacrylamide gel electrophoresis (BN-PAGE) was conducted
as mentioned before (Wei et al., 2020). For mitochondrial
supercomplexes, 1% digitonin (Sigma-Aldrich) was used to dissolve the
membrane protein from patient and controls, then 3.5%-16% gradients
polyacrylamide gel to separate the supercomplexes. For mitochondrial
complexes, 1% dodecyl maltoside (DDM, Sigma-Aldrich) was used and then
3%-11% gradient polyacrylamide gel to separate the complexes. The
proteins were transferred on the PVDF membrane (Bio-Rad, USA), blocked
with 5% no-fatty acid milk (Mengniu, China), incubated with the first
and second antibodies, and detected the protein signals. The following
antibodies were used: anti-Grim19 (1:1000, Sigma-Aldrich), anti-SDHA
(1:3000, Sigma-Aldrich), anti-UQCRC2 (1:2000, Sigma-Aldrich),
anti-MT-COI (1:2000, Sigma-Aldrich), anti-ATP5A (1:3000, Sigma-Aldrich),
anti-NDUFB6 (1:2000, Sigma-Aldrich), anti-NDUFS3 (1:2000,
Sigma-Aldrich), anti-TOM70 (1:2000, Proteintech, China), anti-mouse IgG
HRP linked (1:2000,
Cell Signaling Technology, USA),
anti-rabbit IgG HRP linked (1:2000,
Cell Signaling Technology),
anti-mouse IgG, AP (1:2000,
Cell Signaling Technology).