NF kappa B translocation in human microvessel endothelial cells using a four-compartment subcellular protein redistribution assay

Protein distribution profiles may be used to characterize both physiologica l and pathophysiological cellular changes, but rigorous biochemical assays for measuring such movements are lacking. This paper reports on a protein r edistribution assay that combines reversible metal chelate-based total prot ein detection with a four-fraction subcellular detergent fractionation proc edure. 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 s eparated electrophoretically and electroblotted or slot-blotted onto PVDF m embranes without electrophoretic separation. A key feature is that total pr otein is measured and analyzed directly on the resultant PVDF membrane, usi ng a Ferrozine/ferrous metal-chelate stain, without the added step of a pri or solution-phase protein assay. As a result, factors that may adversely af fect NF kappaB quantification, such as saturation of the solid-support memb rane, are rigorously evaluated and controlled. Following removal of the Fer rozine/ferrous total protein stain, NF kappaB distribution is determined vi a standard immunodetection procedures. This assay reveals a new level of co mplexity regarding NF kappaB distribution and translocation. NF kappaB is s hown to translocate from the cytosol to the membrane/organelle and cytoskel etal/DNA fractions, whereas trace levels of MF kappaB are observed in the n uclear (envelope and associated) fraction. Dose-curve analysis reveals that the response is initiated at 10 U/mI of TNF-alpha, plateaus at approximate ly 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 r esponse at approximately 30 min, after which the distribution begins to ret urn to baseline. The assay should provide a valuable tool for rapid evaluat ion and mechanistic studies of NF kappaB redistribution. (C) 2000 Elsevier Science B.V. All rights reserved.