Fig 1: Muscle-specific changes in mitochondrial DNA (mtDNA) abundance (top) and mitochondrial protein expression (bottom) in adult female C57BL/6j mouse medial gastrocnemius (MG) and tibialis anterior (TA) muscles after long-term voluntary aerobic training. The mtDNA-to-nuclear DNA (nDNA) ratio was compared between sedentary (SED; n = 6) and 5-week-trained mice (EX5; n = 6) using mitochondrial genes cytochrome c oxidase subunit I (COX1) and ATP synthase subunit 6 (ATP6) and the nuclear housekeeping gene, ß2 microglobulin (B2M). Expression of MOTS-c and COX1 proteins also were compared between groups; representative protein and ponceau-stained membranes as a loading control are shown. Values show mean ± S.E. *Statistically different from SED (p < 0.05).
Fig 2: COXIV and ATP6 protein levels in the patient and control. (a) COXIV and ATP6 protein levels were determined with Western blots. (b) The density of each band was quantified with ImageJ. Ac-tubulin was used as the loading control. The results were expressed as the mean ± SD of three independent experiments. Data were analyzed with spss 18.0 software. *p < 0.05, **p < 0.01. (c) COXIV and ATP6 protein expressions were determined by immunofluorescence assay. Multiple images were taken, and representative images are presented. Scale bar: 10 µm
Fig 3: Western blot analysis of mitochondrial proteins. (A) Five micrograms of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with nine respiratory complex subunits in mutant, control cybrid cell lines and HUVECs with TOM20 as a loading control. ND1, ND3, ND4, ND5 and ND6, indicate subunits 1, 3, 4, 5 and 6 of the reduced nicotinamide-adenine dinucleotide dehydrogenase; CYTB, apocytochrome b; CO2, subunit II of cytochrome c oxidase; ATP6 and ATP8, subunit 6 and 8 of the H+-ATPase. (B) Quantification of total mitochondrial protein levels. The levels of mitochondrial proteins in HUVEC and six cybrid cell lines were determined as described elsewhere (22–24). The values for the mutant cybrid cell lines are expressed as percentages of the values for the control cell lines. The calculations were based on three independent determinations. (C) Quantification of levels of 9 polypeptides. The levels of ND1, ND3, ND4, ND5, ND6, CYTB, CO2, ATP6 and ATP8 in HUVEC and six cybrid cell lines were determined as described elsewhere (22–24). Graph details and symbols are explained in the legend to Figure 3.
Fig 4: Mitochondrial RNA processing and polyadenlyation for MT-ATP6.(A) Schematic of the polycistronic heavy-strand RNA transcript highlighting the RNA processing for MT-CO2, ATP6, and CO3. The processing between the ATP6 and CO3 CDS is not mediated by either RNase P or Z. The mechanism of this catalysis is currently unknown. ATP6 and CO2 both encode a stop codon in the CDS. (B) Deep sequencing analysis of the 3' end of the processed ATP6 mRNA from cultured fibroblasts with the indicated genotypes. (C) Total nonstop mRNAs for ATP6 and CO2 generated by truncated polyadenylated transcripts. (D) Deep sequencing analysis of the 3' end of the processed ATP6 mRNA from biologically independent healthy controls taken from cultured fibroblasts and myoblasts and human skeletal muscle biopsies. (E) Total nonstop mRNAs for ATP6 and CO2 generated by truncated polyadenylated transcripts from the samples in (D).
Fig 5: Western blot analysis of mitochondrial proteins. (a) 20 µg of total cellular proteins from various cybrid cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with respiratory complex subunits in mutant and control cells with VDAC as a loading control. (b) Quantification of 6 respiratory complex subunits. The levels of ND6, ND4, ATP6, CO2, and ND1 in two mutant cell lines and two control cybrid cell lines were determined. The error bars indicate two standard errors of the means. *P < 0.05 and **P < 0.01 indicate the significance, according to the t-test, of the differences between mutant and control cybrid cell lines.
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