Ab203832

As previously described (Riessland et al., 2019 ), 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 15min 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 hours. 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).