NFkappaB translocation in human microvessel endothelial cells using a four-compartment subcellular protein...

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.