Mitoprofile

Fibroblast cells were grown in DMEM medium supplemented with glutamax (446 mg/l), 10% fetal calf serum, 50μg/ml uridine and 1mM sodium pyruvate under standard conditions. Cells were fused with 143B rhoo cells as previously described [63 (link)]. Several clones were isolated and the resulting cybrid cells were subsequently expanded. Biochemical assays were performed on isolated mitochondria and/or permeabilized cells. Western blot analysis of a patient’s muscle biopsy sample and Blue Native gel analysis (BNG) of a patient’s fibroblast were performed as previously described [63 (link)]. We used “MitoProfile” antibody mix (Total OXPHOS human WB antibody cocktail, Abcam) for muscle lysates. For fibroblast lysates we used antibody GRIM19 (Abcam), which corresponds to the mitochondrial complex I subunit NDUFA13.

For the immunodetection of respiratory complexes, 20 µg of total protein samples from oral mucosa were fractionated in 12% polyacrylamide gel with running buffer: twenty-five millimolar Tris, 190 mM glycine, 0.1% SDS, pH 8.3 was transferred onto a polyvinyl difluoride (PVDF) membrane using a wet transfer tank with the transfer buffer: twenty-five millimolar Tris, 190 mM glycine, 20% methanol, pH 8.3. The PVDF membrane was blocked for 1 h with 3% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) buffer; 20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20, at room temperature. Then, the PVDF membrane was incubated overnight at 4 °C with MitoProfile^® Total Oxidative Phosphorylation (OXPHOS) Human Western Blot (WB) Antibody Cocktail (ab110411 Abcam) used at 1/200 dilution in 1% BSA in TBST. The blot was rinsed three times for 5 min with TBST buffer. Primary antibodies were detected with the secondary antibodies: Horseradish peroxidase (HRP)-conjugated anti-mouse Immunoglobulin G (IgG) used at 1/300 in 1% BSA in TBST for 1 h at room temperature and the Chemiluminescent Western Blot Detection (ThermoFisher Scientific).