Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System

Figures

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  Figure 1. RT-PCR amplification of DMD transcript in human skeletal muscle. (A) Schematic description of Dp71 mRNA. The exon structure of Dp71 mRNA is shown. Boxes indicate exons. The shaded exon indicates Dp71-specific exon 1 (Exon G1). Numbers in boxes indicate exon number. Brackets and numbers under the bracket indicate the amplified fragment and exon number, respectively. Parenthesis indicates the size of the amplified fragment; (B) amplification of Dp427m and Dp71 mRNA: Dp427m and Dp71 mRNAs were RT-PCR amplified from human skeletal muscle total RNA. Electropherograms of amplified products are shown. The fragment extending from exon M1 to exon 8 was amplified as an expected size product (683 bp) (Dp427, M1-8). The fragment from exon G1 to exon 66 was also amplified as an expected size product (448 bp) (Dp71, G1–66). Full-length Dp71, from exon G1 to exon 79 was also amplified, yielding a single product of expected size (1868 bp) (Dp71, G1–79). Three fragments of Dp71, exon G1 to exon 66 (Dp71, G1–66); exon 67 to exon 72 (Dp71, 67–72), and exon 70 to exon 79 (Dp71, 70–79) were amplified and sequenced. As an internal control, the GAPDH cDNA was also amplified (GAPDH). Mk refers to size…

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  Figure 2. Detection of over-expressed Dp71 by the Simple Western. The Dp71 encoding plasmid was transfected into HEK293 cells and cell lysates were analyzed by the Simple Western using an antibody against the C-terminal of dystrophin. (A) Simple Western image pseudo-gel views of the molecular weight ladder and cell lysates are shown. A band near the 66 kDa molecular weight marker was identified in cells with the Dp71-expressing plasmid, but not in non-transfected cells; (B) electropherogram traces of cell lysates are shown. The Dp71 peak was not visible in non-transfected cells (−Dp71), whereas a band near the 66 kDa marker was observed in transfected HEK293 cells (+Dp71).

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  Figure 3. Detection of Dp71 in rhabdomyosarcoma cells. (A) Detection and suppression of Dp71 in CRL-2061 cells. CRL-2061 cell lysates were analyzed by the Simple Western. Electropherogram traces of the lysate are shown. A Dp71 peak was observed near the 66 kDa marker (−AO). The identity of this peak as Dp71 was confirmed by disrupting Dp71 mRNA reading frame by transfection of the antisense oligonucleotides AODys75-3. The transfected cells were harvested and analyzed. The Peak height of Dp71 was much lower (+AO), indicating that disruption of the Dp71 mRNA reading frame reduced Dp71 production. β-actin was used as the loading control (bottom panel); (B) effect of AO that induces DMD exon 75 skipping. AODys75-3 against the exonic splicing enhancer sequence within DMD exon 75 was synthesized and its ability to induce exon 75 skipping was assessed. RT-PCR products encompassing exons 73 to 77 are shown. In non-transfected CRL-2061 cells, normal product was obtained (−AO). In AODys75-3 transfected cells, the normal sized band was barely visible and the band appeared as a small size band having a deletion of exon 75 (+AO), indicating that AODys75-3 induced 75 skipping. The exon structure of the…

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  Figure 4. Dp71 in skeletal muscles from normal and mdx mice. (A) Skeletal muscles of C57BL/6 mice and DMDmdx mice were analyzed three times by the Simple Western, with the results shown as electropherogram traces. The Dp71 peak recognized by anti-dystrophin antibody, ab15277, was present in all three samples from normal and DMDmdx mice (top). Simultaneously, vinculin was analyzed as a loading control in the same samples (bottom). The arrows indicate the Dp71 and vinculin peaks; (B) the ratio of Dp71 to vinculin in samples from normal and DMDmdx mice. Dp71 to vinculin ratio was calculated from the AUC of each peak. Bars represent means ± S.EM. * p < 0.05 compared with normal mice.

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  Figure 5. Dp71 in human skeletal muscle. (A) Dose-responsive changes in the Dp71 peak. Human skeletal muscle lysates (SK1) were gradient diluted and analyzed by the Simple Western using an antibody against the C-terminal domain of dystrophin, ab15277. A peak corresponding to Dp71 was identified near 66 kDa marker, with peak height decreasing as loading protein decreased (arrow); (B) Dp71 not recognized by antibodies to the dystrophin rod-domain. The specificity of the Dp71 peak was examined with several primary antibodies, with results shown as electropherogram traces of the Simple Western. The peak was recognized by antibody against the C-terminal domain of dystrophin (ab15277) (shaded peak), but not by antibodies against the rod domain of dystrophin, including antibodies against exon 43 (MANDYS106) and exon 58 (MANEX58). The arrows indicate the Dp71 peaks.

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  Figure 6. Levels of Dp71 protein expression in human skeletal muscles. Five samples of human skeletal muscles (SK1-SK5) were analyzed using the Simple Western system with antibody against the C-terminal domain of dystrophin (ab15277), and anti-vinculin antibody as a loading control. Electropherogram traces of are shown (A). The Dp71 peaks (arrows) in the samples were detected at the same location but at different heights. The Dp71/vinculin ratio (mean ± S.E.M.) was calculated in each sample (B) and found to range between 0.05 and 0.49. Numbers under the columns indicate sampling age (years). M and F indicate male and female, respectively.