Fig. 4. Aac2 oligomerization is specific.

(A) WT mitochondria (100 μg), untreated (−) or instead incubated with CATR (40 μM) or BKA (10 μM), were solubilized with digitonin [1.5% (w/v)] or increasing amounts of DDM [0.32 and 0.64% (w/v)], resolved by 6 to 16% BN-PAGE, and immunoblotted for Aac2 (top), complex III (Rip1, middle), or complex IV (Cox4, bottom). Aac2₁, Aac2 monomer; III₂IV₂-Aac2 and III₂IV-Aac2, Aac2 associated with large and small RSCs, respectively (n = 4). (B) WT mitochondria (250 μg), preincubated with CATR (40 μM) as listed, were solubilized with digitonin [1.5% (w/v)] or increasing amounts of DDM [↓ = 0.32% and ↑ = 0.64% (w/v)], and FLAG-Aac2 immunoprecipitated using anti-FLAG resin. The presence of copurified HA-Aac2 and subunits of complexes III (Cor1, Cor2, Rip1, and Qcr6) and IV (Cox1 and Cox4) was determined by immunoblotting; Atp1, Atp2, Om45, and Kgd1 served as controls. *, nonspecific bands. Four percent of input (mitochondria) and unbound (flow through following FLAG immunoprecipitation) was analyzed (n = 3).