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

• Published in: International journal of molecular sciences 2018-05, Vol.19 (6), p.1546
• Main Authors: Kawaguchi, Tatsuya, Niba, Emma Tabe Eko, Rani, Abdul Qawee Mahyoob, Onishi, Yoshiyuki, Koizumi, Makoto, Awano, Hiroyuki, Matsumoto, Masaaki, Nagai, Masashi, Yoshida, Shinobu, Sakakibara, Sachiko, Maeda, Naoyuki, Sato, Osamu, Nishio, Hisahide, Matsuo, Masafumi
• Format: Article
• Language: English
• Subjects: Animals; Antisense oligonucleotides; Assaying; Automation; Cell Line, Tumor; Cells, Cultured; Complementary DNA; Dp71 protein; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - genetics; Dystrophin - metabolism; Gene expression; Glucose; HEK293 Cells; Humans; Isoforms; Mice; Mice, Inbred C57BL; Muscle, Skeletal - metabolism; Muscles; Muscular dystrophy; Musculoskeletal system; Proteins; Rhabdomyosarcoma; Skeletal muscle; Vinculin; Vinculin - genetics; Vinculin - metabolism; Western blotting
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms19061546

Dystrophin Dp71 is one of the isoforms produced by the gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle. Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMD mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively. Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression. Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.