Western blot analysis of respiratory-deficient mitochondrial cybrids

Copyright information:Taken from "Production of mitochondrial cybrids containing naturally occurring pathogenic mtDNA variants"Nucleic Acids Research 2006;34(13):e95-e95.Published online 2 Aug 2006PMCID:PMC1540737.© 2006 The Author(s) Mitochondrial protein was isolated from 143B.ρ (ρ), parental (Con) and the two mitochondrial cybrids 1.12 and 1.14. () Fifteen micrograms were subjected to western blot analysis using antibodies against complex I (nuclear-encoded 39 kDa or mtDNA-encoded ND6), complex III (nuclear-encoded core 2) and complex IV (mtDNA-encoded COX1 and COX2). All mtDNA-encoded polypeptides are substantially depleted in clone 1.12, consistent with a general translation defect. Clone 1.14 harboured lower steady-state levels of COX proteins but were still substantially higher than either the 143B cell line lacking mtDNA (ρ) or 1.12. Equal loading of mitochondrial protein was confirmed with an antibody specific to the outer membrane protein, porin. () A total of 100 μg of protein was subjected to a non-denaturing gel before visualization of intact complex I and complex IV with anti-39 kDa and COX2 antibodies, respectively. Clone 1.12 is depleted for both holoenzymes, whereas 1.14 has a specific decrease in complex IV. Minor subcomplexes of complex I can be seen in mitochondria isolated from 1.12.