Ab110249

Protein samples were extracted with non-reducing Laemmli buffer without bromophenol blue and quantified by the BCA assay. Extracts were then loaded onto 10% standard polyacrylamide gel electrophoresis after adding 5% 2-mercaptoethanol, and transferred to nitrocellulose membranes or PVDF membranes for BN-PAGE. The following antibodies were used: monoclonal anti-HIF-1α antibody (#MAB1536; R&D Systems), monoclonal anti-NDUFS4 antibody (ab87399; Abcam), monoclonal anti-NDUFS2 antibody (ab110249; Abcam), anti-NDUFB6 antibody (16037-1-ap, Proteintech), anti-ubiquinol-cytochrome c reductase core protein I antibody (ab110252; Abcam), anti-PINK1 (sc-33796, Santa Cruz Biotechnology) and monoclonal anti-α-tubulin antibody (T6199, Sigma). Antibody binding was detected by chemiluminescence with species-specific secondary antibodies labelled with horseradish peroxidase (HRP), and visualized on a digital luminescent image analyzer (Fujifilm LAS-4000).

Protein extraction from cybrids for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting was carried out as described previously (Ji et al., 2020 (link)). Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, United States). The protein extracts were resolved in a 10% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk for 1 h and incubated with the following primary antibodies at 4°C overnight: anti-MT-CO3 (cat #ab110259; Abcam, Cambridge, United Kingdom) and VDAC (cat #4661; Cell Signaling Technology, Danvers, MA, United States); the latter served as a loading control. Blue native (BN)-PAGE was performed with mitochondrial proteins isolated from cybrids as described previously (Delmiro et al., 2013 (link)), using antibodies against MT-COXIV (ab202554; Abcam), SDHA (ab14715; Abcam), VDAC (cat#4661; Cell Signaling Technology), UQCRC2 (ab14745; Abcam), NDUFS2 (ab110249; Abcam), and ATPB (ab14730; Abcam). The in-gel activity (IGA) of CIV was performed as described previously (Jha et al., 2016 (link)). Briefly, after running the native PAGE, incubate the gel in 20 ml of complex IV substrate. Appearance of brown bands is indicative of CIV activity.

ATP5F1 (ab117991, Abcam, 1:1000), β-Actin (ab8224, Abcam, 1:1000), COX IV-Alexa Fluor 488 (4853 S, Cell Signaling Tech [CST], 1:200), EMRE (A300-BL19208, Bethyl, 1:1000), FLAG HRP (A8592, Sigma, 1:1000), FLAG magnetic beads (M8823, Sigma), FOXRED1 (sc-377264, Santa Cruz, 1:1000), GAPDH (2118 S, CST, 1:1000), GFP (ab290, Abcam, BN-PAGE: 1:1000), goat anti-mouse Alexa Fluor 555 (A21422, ThermoFisher, 1:100), goat anti-rabbit Alexa Fluor 488 (A32731, ThermoFisher, 1:100), HA (3724 S, CST, 1:800), HA HRP (12013819001, Sigma, 1:1000), MCU (14997 S, CST, WB 1:1000, ICC 1:100, DuoLink 1:200), MICU1 (12524 S, CST, 1:1000), MICU1 (HPA037480, Sigma, 1:1000), MTCO1 (ab14705, Abcam, 1:100), NDUFA13 (ab110240, Abcam, 1:1000), NDUFB10 (ab196019, Abcam, 1:1000), NDUFS2 (ab110249, Abcam, WB 1:1000, ICC 1:100, DuoLink 1:200), NDUFS3 (ab177471, ICC 1:200), NDUFS4 (ab137064, WB 1:1000), NDUFS4 (PA5-21677, ThermoFisher, BN-PAGE: 1:300), Oct4 (ab19857, Abcam, 1:200), ROMO1 (TA505580, Origene, 1:1000), Sox2 (5024, CST, 1:200), TOM20 (42406 S, CST, BN-PAGE: 1:800), VDAC1 (ab14734, Abcam, 1:1000), Vimentin (ab92547, Abcam, 1:1000), ZO-1 (33-9100, ThermoFisher, 1:200).