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Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells

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The Nuclear Envelope

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1411))

Abstract

Nuclear protein import and export assays in permeabilized cells have been instrumental for the identification of transport factors and for the molecular characterization of nucleocytoplasmic transport pathways. Our original assay to quantitatively analyze CRM1-dependent export was based on stably transfected cells expressing GFP-NFAT. We now present a simplified version of the assay using transiently transfected cells expressing GFP-NFAT or GFP-snurportin1 as a fluorescent export cargo and mCherry-emerin as a marker protein for transfected cells. CRM1- and Ran-dependent export is recapitulated in digitonin-permeabilized cells and quantified by flow cytometry. The assay should be applicable to other combinations of cargo and marker proteins.

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Author information

Authors and Affiliations

  1. Faculty of Medicine, Institute of Molecular Biology, University of Göttingen, Humboldtallee 23, 37073, Göttingen, Germany

    Ralph H. Kehlenbach & Sarah A. Port

Authors
  1. Ralph H. Kehlenbach
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  2. Sarah A. Port
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Corresponding author

Correspondence to Ralph H. Kehlenbach .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, University of Leicester, Leicester, United Kingdom

    Sue Shackleton

  2. Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Philippe Collas

  3. Wellcome Trust Centre for Cell Biol, University of Edinburgh, Edinburgh, United Kingdom

    Eric C. Schirmer

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Kehlenbach, R.H., Port, S.A. (2016). Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_30

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  • DOI: https://doi.org/10.1007/978-1-4939-3530-7_30

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3528-4

  • Online ISBN: 978-1-4939-3530-7

  • eBook Packages: Springer Protocols

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