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Methods Mol Biol. 2016;1411:489-501. doi: 10.1007/978-1-4939-3530-7_30.

Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells.

Author information

1
Faculty of Medicine, Institute of Molecular Biology, University of Göttingen, Humboldtallee 23, 37073, Göttingen, Germany. rkehlen@gwdg.de.
2
Faculty of Medicine, Institute of Molecular Biology, University of Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.

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.

KEYWORDS:

CRM1; Digitonin; Export; Flow cytometry; NFAT; Nuclear transport; Nucleus; Ran; Snurportin 1

PMID:
27147061
DOI:
10.1007/978-1-4939-3530-7_30
[Indexed for MEDLINE]

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