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. 2013 Jan 29;288(12):8321–8331. doi: 10.1074/jbc.M112.422220

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 8. — hCOA3 interacts with COX structural subunits and complex I. A, two-dimensional BN/SDS-PAGE of hCOA3-FLAG-overexpressing HeLa cells. S1, S2 and S3 indicate the three COX subassemblies, S4 indicates the COX monomer, and CIII+IV indicates supercomplex III+IV. B, immunoprecipitation using anti-FLAG M2 antibodies in control (pIRESpuro2) and hCOA3-FLAG-overexpressing (hCOA3-FLAG) mitochondria. Proteins were extracted with 1% DDM. 10% of the corresponding volume of input, unbound, and wash fractions was loaded on the gels. C, proteins were extracted from isolated mitochondria from HeLa cells and HeLa cells overexpressing hCOA3-FLAG with 1% digitonin. Extracts were incubated with protein A-Sepharose beads and anti-FLAG M2 antibodies where indicated. Beads and supernatant were recovered by centrifugation. Samples were analyzed by Western blotting.