Ab110411

Protein concentrations of the LV tissues were determined by the BCA protein assay kit (Thermo Fisher, Erembodegem, Belgium). Western blot was performed as previously described^{40 (link)}. Briefly, equal amounts of proteins (15 µg) were separated on a 12% SDS-PAGE gel with a mini protean 3 electrophoresis system (Bio-rad Laboratories, Temse, Belgium), transferred to a polyvinylidene fluoride (PVDF) membrane and subsequently, blocked for 2 h with 5% milk in Tris-buffered solution containing 0.1% Tween-20 (TBS-T) followed by incubation overnight at 4 °C in the presence of a specific endothelin-1 antibody (1/2500, Abcam, ab117757, Cambridge, United Kingdom), NOX2 antibody (1/2500, Abcam, ab31092, Cambridge, United Kingdom) or an OXPHOS antibody (1/1000, Abcam, ab110411, Cambridge, United Kingdom). Horseradish peroxidase-conjugated secondary antibodies (DAKO, Belgium) at a dilution of 1/2000 were used. Both primary and secondary antibodies were diluted in 5% milk-TBS-T. Visualization was performed with the enhanced chemiluminescence (ECL) technique using the Pierce ECL Plus western Blotting Substrate (Thermo Fisher, Erembodegem, Belgium). Data were normalized to β-actin protein levels.

For both pre and post-intervention muscle biopsies, it was ensured that participants were fasted state (10–12 h) and refrained from physical activity for 72 h prior to collection. Final training sessions were staggered across all groups to ensure accurate timing of the 72 h post-training muscle samples. After administration of a local anaesthetic (2% plain Lignocaine) a muscle biopsy was obtained from the lateral portion of the m. vastus lateralis of each participant. Using a 5-mm Bergstrom needle an ~100 mg sample was obtained, blotted on filter paper, removed of fat and connective tissue, frozen in liquid nitrogen and stored at -80°C until further analysis. Samples were analyzed for total protein content of PGC-1α (Calbiochem ST1202), nuclear respiratory factor (NRF)1 (Abcam ab34682), NRF2 (Santa Cruz sc-22810), mitochondrial transcription factor (Tfam; Abcam ab47517), mitochondrial complex I-V (Mitoprofile Human Total OXPHOS antibody cocktail, Mitosciences ab110411), myocyte enhancer factor 2A (MEF2A; Abcam ab87975), SIRT 1 (Cell Signaling 8469), GLUT4 (Millipore CBL242), p53 (Cell Signaling 2527), AKT (Cell Signaling 9272) and α-tubulin (Cell Signaling 2125), which was used as a loading control.

Cell pellets were lysed in RIPA buffer containing 1× Halt^TM protease inhibitor cocktail (78437, Thermo) and supernatants collected after 10 min centrifugation at 12000 × g for SDS-PAGE. Total proteins were transferred to PVDF membranes and blotted with the antibodies against FIS1 (ab71498, Abcam), MFN1 (ab104274, Abcam), β-Actin (ab8227, Abcam), Caspase-3 (9665, Cell Signaling), Caspase-8 (sc-5263, Santa Cruz), Caspase-9 (sc-8355, Santa Cruz), HSP70 (TA309356, Origene) and Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam).