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  • 标题:Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies
  • 本地全文:下载
  • 作者:Julia Maier ; Bjoern Traenkle ; Ulrich Rothbauer
  • 期刊名称:Scientific Reports
  • 电子版ISSN:2045-2322
  • 出版年度:2015
  • 卷号:5
  • DOI:10.1038/srep13402
  • 出版社:Springer Nature
  • 摘要:Vimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.
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