Figure 4.
MPV17L2 distribution overlaps with mtDNA on iodixanol gradients, whilst in cells lacking mtDNA MPV17L2 is not detectable. (A) Mitochondrial 1000 gmax supernatants from HEK293T cells were fractionated on 20–42.5% iodixanol gradients, the DNA content of each fraction was determined by ethidium bromide staining after agarose gel electrophoresis. Proteins were separated on 4–12% gradient, or 12 or 16% linear SDS-PAGE, proteins were detected by immunoblotting with the indicated antibodies (see text for details). (B) Immunoblots of total cellular protein from HEK293T cells with (ρ+) and without (ρ0) mtDNA. ATAD3, ATPase family AAA domain-containing protein 3; COX2, Cytochrome c Oxidase subunit 2; Core1, nuclear subunit Complex III; HSP60, Heat Shock Protein 60; MRPL3, MPV17, homologous protein to MPV17L2; Mitochondrial Ribosomal Large Subunit Protein 3; MRLS18B, Mitochondrial Ribosomal Small Subunit Protein 18B; MRLS27, Mitochondrial Ribosomal Small Subunit Protein 27; TFAM, mitochondrial Transcription Factor A. (C) HEK293T cells were treated with or without 100 ng/ml ethidium bromide (EB) for 72 h. Mitochondrial lysates (ML) (see ‘Materials and Methods’ section) were fractionated on 20–42.5% iodixanol gradients. DNA or protein was extracted after fraction collection and analysed by Southern hybridization (mtDNA) and immunoblotted with the indicated antibodies.