title: Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells.
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.
authors.affiliation: Faculty of Medicine, Institute of Molecular Biology, University of Göttingen, Humboldtallee 23, 37073, Göttingen, Germany. rkehlen@gwdg.de.
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Kehlenbach
authors.affiliation: Faculty of Medicine, Institute of Molecular Biology, University of Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Port
mesh_terms: D021581:Active Transport, Cell Nucleus; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004072:Digitonin / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
chemical_list: D004072:Digitonin; D007202:Indicators and Reagents; D028884:Karyopherins; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C092710:emerin
keywords: CRM1; Digitonin; Export; Flow cytometry; NFAT; Nuclear transport; Nucleus; Ran; Snurportin 1