Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System.
Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System.
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DOI：
10.3390/ijms19061546
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发表时间：
2018-05-23
影响因子：
5.6
通讯作者：
Matsuo M
中科院分区：
文献类型：
--
作者：
Kawaguchi T;Niba ETE;Rani AQM;Onishi Y;Koizumi M;Awano H;Matsumoto M;Nagai M;Yoshida S;Sakakibara S;Maeda N;Sato O;Nishio H;Matsuo M
背景：肌营养不良蛋白 Dp71 是杜氏肌营养不良症 (DMD) 患者中发生突变的 DMD 基因产生的亚型之一。尽管 Dp71 普遍表达，但在正常骨骼肌中尚未检测到。本研究旨在评估 Dp71 在人类骨骼肌中的表达。方法：人骨骼肌RNA和组织是商业获得的。小鼠骨骼肌取自正常小鼠和 DMDmdx 小鼠。 Dp71 mRNA 和蛋白质分别通过逆转录 PCR 和自动毛细管蛋白质印迹分析系统 Simple Western 测定。分别使用表达 Dp71 的质粒或反义寡核苷酸来过表达或抑制 Dp71。结果：全长 Dp71 cDNA 通过 PCR 扩增为人骨骼肌 RNA 的单一产物。大约。通过在HEK293细胞中过表达Dp71，或在横纹肌肉瘤细胞中用反义寡核苷酸抑制Dp71表达，将Simple Western检测到的70 kDa蛋白峰确定为Dp71。 Simple Western 检测在正常小鼠和 DMD 小鼠的骨骼肌中均检测到了 Dp71。在人类骨骼肌中，也检测到了 Dp71。人类骨骼肌样本中 Dp71 与纽蛋白的比例差异很大，表明 Dp71 表达水平不同。结论：使用高灵敏度毛细管蛋白质印迹系统检测人骨骼肌中的 Dp71 蛋白。
Background: Dystrophin Dp71 is one of the isoforms produced by the DMD 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. Methods: Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMDmdx 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. Results: 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. Conclusions: Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.
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DOI：
10.1016/j.bbrc.2017.08.108
发表时间：
2017-10-21
影响因子：
3.1
作者：
Fujimoto, Takahiro;Yaoi, Takeshi;Itoh, Kyoko
通讯作者：
Itoh, Kyoko
影响因子：
120.1
作者：
Neef, Daniel W.;Jaeger, Alex M.;Thiele, Dennis J.
通讯作者：
Thiele, Dennis J.
影响因子：
5.2
作者：
Moizard, MP;Toutain, A;Moraine, C
通讯作者：
Moraine, C
影响因子：
3.5
作者：
Lee T;Awano H;Yagi M;Matsumoto M;Watanabe N;Goda R;Koizumi M;Takeshima Y;Matsuo M
通讯作者：
Matsuo M
影响因子：
3.5
作者：
LEDERFEIN, D;YAFFE, D;NUDEL, U
通讯作者：
NUDEL, U