Mitochondrial Complex Assessment by Western Blot

As previously described (Riessland et al., 2019 (link)), for the assessment of mitochondrial complexes, cell lysates were prepared using the NativePAGE Sample Prep Kit (Life Technologies) and solubilized with 1% digitonin. A BCA assay (Thermo Scientific) was used to determine the protein concentrations as described above. Equal amounts of protein were then loaded onto a NativePAGE Novex 4%–16% Bis‐Tris Protein Gel (Life Technologies). A wet blotting method was used to transfer the proteins onto a PVDF membrane (Life Technologies, 0.45 μm), the membrane was then incubated with 8% acetic acid for 15 min and washed with methanol and water before being blocked with 5% BSA in 20 mM Tris, 150 mM NaCl, and 0.1% (w/v) Tween 20, pH 7.5, for 2 h. The membrane was subsequently immunoblotted with the respective primary antibody at 4°C overnight. The primary antibodies used were NDUFA9 (a CI Subunit) (1/2500 by vol; ab14713: Abcam), UQCRC2 (a CIII Subunit) (1/2500 by vol; ab203832: Abcam), or MT‐CO2 (a CIV Subunit) (1/2500 by vol; ab110258: Abcam). Primary antibodies were detected using either HRP‐linked donkey anti–rabbit IgG (GE Healthcare, #NA934V; 1:10,000) or HRP‐linked sheep anti–mouse IgG (GE Healthcare, #NA931V, 1:10,000) together with Western Lightning Plus‐ECL (Perkin Elmer, #NEL105001EA).