Pioglitazone (PGZ) decreased the amount of prohibitin, NDUFAF1, and FOXRED1 associated with mitochondrial complexes and joined subunits NDUFA9, NDUFB6, and NDUFA6 of complex I. Mouse mitochondria were treated with PGZ, proteins were separated by either blue native polyacrylamide gel electrophoresis (BN-PAGE) (A) or sodium dodecyl sulfate (SDS)/BN-PAGE (B) and analyzed by western blot using antibodies against prohibitin, NDUFAF1, or FOXRED1. C, control, untreated proteins. P, PGZ treated proteins. (C) Mouse liver mitochondria were treated with 10 μM [3H]PGZ for 30 minutes. Mitochondrial complexes were separated in parallel on three one-dimensional BN-PAGE gels. One gel was used to localize [3H]PGZ by autoradiography, the second gel was utilized to analyze complex I by western blot, and the third was employed to determine complex I in-gel activity. (D) Mitochondrial complexes were separated in parallel on two SDS/BN-PAGE gels. One gel was analyzed by western blot using antibodies against complex I subunits NDUFS1, NDUFV1, NDUFA9, NDUFV2, NDUFB6, NDUFA6, and NDUFA2. The second gel was used to localize [3H]PGZ by autoradiography. (E) Liver mitochondria treated with 10 μM [3H]PGZ for 30 minutes were immunoprecipitated with monoclonal antibody against NDUFA9 (A9), NDUFB6 (B6), NDUFA6 (A6), NDUFV2 (V2), NDUFS1 (S1), core 1 (Cor), and COX1 (COX) as described in the Methods section. Radioactivity was quantitated in the immunoprecipitates. (F) Mitochondrial proteins treated with 10 μM [3H]PGZ for 30 minutes were immunoprecipitated as indicated in (E), electrophoresed in a 10% acrylamide gel, and transferred to a nitrocellulose membrane. Subunits were analyzed by western blot to show specific immunoprecipitation of subunits. Individual bands were excised and their radioactivity was measured. Lane 1, western blot of not immunoprecipitated mitochondrial proteins. Lanes A6, B6, V2, A9, Cor, COX, and S1 western blot of immunoprecipitated NDUFA6, NDUFB6, NDUFV2, NDUFA9, core 1, COX1, and NDUFS1 subunits, respectively.