Background: The mechanism by which tumor necrosis factor (TNF) activates endothelial cells in not well understood. Endothelial cells express both the 55 kd (TNF-R1) and 75 kd (TNF-R2) TNF receptors, and it has been shown that TNF-R2 is expressed predominantly on the cell surface whereas TNF-R1 is localized largely in an intracellular compartment. In order to identify structural features responsible for these distributions and to further elucidate transduction of the TNF signal, we have overexpressed epitope-tagged (FLAG) TNF-R1 and TNF-R2 in human umbilical vein endothelial cells (HUVEC).

Methods: HUVEC were transfected with expression vector constructs of the full length cDNA for either TNF-R1 or TNF-R2. The receptors were epitope-tagged with FLAG on the amino terminus. Green fluorescent protein(GFP) was co-transfected as an independent marker fo transfected EC. The transfections were done using DEAE/dextran. After harvesting, the cells were divided and half were permeabilized. They then underwent immunofluorescent staining and FACS analysis.

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