title: NFkappaB translocation in human microvessel endothelial cells using a four-compartment subcellular protein redistribution assay.
abstract: Protein distribution profiles may be used to characterize both physiological and pathophysiological cellular changes, but rigorous biochemical assays for measuring such movements are lacking. This paper reports on a protein redistribution assay that combines reversible metal chelate-based total protein detection with a four-fraction subcellular detergent fractionation procedure. TNF-alpha stimulated cultured human omental microvessel endothelial cells are fractionated into cytosol, membrane/organelle, nuclear (envelope and associated), and cytoskeletal/DNA compartments. Protein fractions are separated electrophoretically and electroblotted or slot-blotted onto PVDF membranes without electrophoretic separation. A key feature is that total protein is measured and analyzed directly on the resultant PVDF membrane, using a Ferrozine/ferrous metal-chelate stain, without the added step of a prior solution-phase protein assay. As a result, factors that may adversely affect NFkappaB quantification, such as saturation of the solid-support membrane, are rigorously evaluated and controlled. Following removal of the Ferrozine/ferrous total protein stain, NFkappaB distribution is determined via standard immunodetection procedures. This assay reveals a new level of complexity regarding NFkappaB distribution and translocation. NFkappaB is shown to translocate from the cytosol to the membrane/organelle and cytoskeletal/DNA fractions, whereas trace levels of NFkappaB are observed in the nuclear (envelope and associated) fraction. Dose-curve analysis reveals that the response is initiated at 10 U/ml of TNF-alpha, plateaus at approximately 1000 U/ml, and remains essentially constant up to 2000 U/ml. Time-course analysis demonstrates a measurable response as early as 5 min and a peak response at approximately 30 min, after which the distribution begins to return to baseline. The assay should provide a valuable tool for rapid evaluation and mechanistic studies of NFkappaB redistribution.
authors.affiliation: Microvascular Research Laboratory, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Chiang
authors.forename: M J
authors.initials: MJ
authors.lastname: Lim
authors.forename: W F
authors.initials: WF
authors.lastname: Patton
authors.forename: D
authors.initials: D
authors.lastname: Shepro
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D015153:Blotting, Western; D002458:Cell Fractionation; D002478:Cells, Cultured; D003902:Detergents; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005297:Ferrozine / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism*; D009852:Omentum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D003902:Detergents; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D005297:Ferrozine