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. 2007 Sep 20;26(20):4335–4346. doi: 10.1038/sj.emboj.7601861

Figure 7.

Figure 7

Coa1 and Mss51 form similar sized complexes and interact. (A) Extracts of solubilized mitochondria (0.5 mg protein) from COA1-13Myc MSS51-3HA cells were fractionated over Superdex 200 in solubilization buffer (PBS, 0.1% DOC). The fractions were analyzed by immunoblot. (B) Mitochondria isolated from either wild-type (WT), shy1Δ or cox14Δ strains containing genomically tagged COA1 (COA1-13Myc, lanes 1–3) or MSS51 (MSS51-13Myc, lanes 4–6) were solubilized in buffer containing 1.5% digitonin. Lysates were loaded onto a continuous 4–13% gradient gel and protein complexes were separated by BN-PAGE. The distribution of complexes was analyzed by immunoblotting with mouse monoclonal anti-Myc antibody. Monomeric (V1) and dimeric (V2) forms of respiratory chain complex V served as a control and were visualized using antisera against F1-subunit. (C) Mitochondria (0.3 mg protein) from COA1-13Myc or COA1-13Myc MSS51-3HA cells were solubilized in Tris 20 mM pH 7.4, 100 mM NaCl, 1 mM PMSF, 0.1% lauryl maltoside and clarified extracts were immunoprecipitated with rabbit polyclonal HA antiserum and protein A agarose beads. The load representing 5% of the extracts and the entire fraction of the last wash and bead eluate were analyzed by immunoblotting.