Pioglitazone (PGZ) disassembled complex I into four subcomplexes of approximately 600, 400, 350, and 250 kDa, respectively. (A) Mitochondrial complexes were treated with 10 μM PGZ as described in Methods and Figure 2A, separated on one-dimensional 5% to 15% blue native polyacrylamide gel electrophoresis (BN-PAGE), and analyzed by western blot using antibodies against subunits NDUFA9, NDUFA6, NDUFV1, NDUFV2, NDUFS1, NDUFS7, NDUFS3, NDUFB6, NDUFB8, NDUFC2, MTND4, and MTND6. Molecular weight was determined by the mobility of fully assembled complex I (980 kDa), complex V (650 kDa), complex III (500 kDa), complex IV (200 kDa), and complex II (130 kDa). (B) Mouse liver mitochondria (30 μg) were exposed to 10 μM PGZ for 30 minutes and separated in the first dimension using a 5% to 15% BN-PAGE gel and in the second dimension using sodium dodecyl sulfate (SDS)-PAGE. Subunits were identified by western blot using specific antibodies against subunits NDUFA9, NDUFA6, NDUFV1, NDUFV2, NDUFS1, NDUFS7, NDUFS3, NDUFB6, NDUFB8, NDUFC2, MTND6, and MTND4 of complex I, as well as antibodies against subunits SDHA of complex II, core 2 of complex III, COX4 of complex IV, and subunit ATP5A1 of complex V. These results are representative of three separate experiments.
García-Ruiz et al. BMC Biology 2013 11:88 doi:10.1186/1741-7007-11-88