Coxiv

For protein extraction, transfected HEK293 cells with hMfsd7c, mMfsd7c, hMfsd7c mutants were lysed with RIPA buffer, respectively. For validation of deletion of Mfsd7c in mice, micro-vessels from PBS-perfused brains of WT and EcMfsd7c-KO mice were isolated, homogenized and lyzed with RIPA buffer. For Western blot analysis of hMfsd7c expression in yeasts, 1 mL of yeast culture at OD₆₀₀ = 1 from WT, Hnm1 mutant yeasts or transformed yeasts was used for total protein extraction. BCA assay was used for total protein quantification. These antibodies were used: VDAC (Cell Signaling, CS4866), CoxIV (Invitrogen, MA5-15686), OPA1 (BD biosciences, 612602), MRPS35 (Protein biotech, 16457), CHKA (Cell Signaling, CS13422S), HMOX1 (Proteintech, 10701-1-AP), NDUFS1 (Proteintech, 12444-1-AP). Western blot analysis was conducted as previously described.^{2 (link)}

Muscle homogenates, purified isolated sarcolemmal vesicles and mitochondrial fractions were analyzed for total protein (BCA protein assay). Five micrograms of denatured protein from each sample were separated by electrophoresis on 7.5% and 12% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. Thereafter, membranes were blocked in 7.5% BSA and probed over night with commercially available antibodies against FAT/CD36 (rats; donated by Dr. Tandon; mice; Santa Cruz), FABPpm (donated by Dr. Calles-Escandon), COXIV (cytochrome c oxidase IV; Invitrogen), Cav-1 (caveolin-1; BD Biosciences), MCT1 (monocarboxylate transporter 1; Abcam), SERCA2 (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; Thermo Scientific), AMPKα total and AMPKα Thr172 phosphorylation (Cell Signaling), ACC (acetyl-CoA carboxylase) and ACC Ser79 phosphorylation (Cell Signaling), extracellular signal-regulated kinase (ERK1/2) total and Thr202/Tyr204 phosphorylation (Cell Signaling). All blots were quantified using enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON) and quantified by densitometry (Alpha Innotech Fluorchem HD2, Fisher Scientific, Ottawa, ON).

The following antibodies were used for this study: β-actin (western blot 1:1000, Enzo Life Science, ADI-905–733), CoxIV (western blot 1 μg/ml, Invitrogen, 459600), EndoG (western blot 1:200, Santa Cruz Biotechnology, sc-26923), Histone H3 (western blot 1:250, Diagenode, cs-135–100), γ-H2AX (immunofluorescence 0.2 μg/ml, Millipore, 05–636), 53BP1 (immunoflourescence 1:100, Abcam, ab36823).