Figure 2 | Scientific Reports

Figure 2

From: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function

Figure 2

143B MCAD knockout (KO) cells also exhibit reduced steady-state levels of OXPHOS complexes and supercomplexes. The ACADM gene was edited in 143B cells using lentiviral-based CRISPR/Cas9. Mitochondria were isolated from 143B control cells (CON) or a 143B MCAD knockout clone (KO) for SDS-PAGE and BN-PAGE Western blot analysis. (A) No mature MCAD protein is detectable in KO mitochondria by SDS-PAGE (the mitochondrial protein VDAC1 is shown as a loading control). BN-PAGE revealed that the steady-state levels of mature OXPHOS complex IV (detected with an anti-COI antibody) (B), complex I (anti-NDUFA9 antibody) (C), the complex III dimer (CIII2) and the CIII2/CIV supercomplex (anti-UQCRC1 antibody) (D) and the CI/CIII2/CIV supercomplex (anti-NDUFA9 antibody) (E) were all significantly reduced in KO mitochondria compared to the control (CON). Steady-state levels of OXPHOS complex V (anti-ATP5A antibody) (F) and the Translocase of the Outer Mitochondrial Membrane (anti-TOMM40 antibody) (G) were not different in KO mitochondria compared to the control (CON). (G) The homotetrameric MCAD complex is detectable in CON, but not MCAD KO mitochondria. Mitochondria were solubilised in TX-100 (B–D) or in digitonin (E–G). Quantitation is relative to the steady-state levels of OXPHOS complex II (anti-SDHA antibody) (n = 3).

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