Enforced expression of IκBmt protein inhibits NF-κB activation in the presence of TRAF6. (A) Expression of Flag-tagged IκBmt, TRAF2, and TRAF6 in retrovirally transduced endothelial cell lines was verified by immunoblotting of total cell extracts using the M5 anti-Flag monoclonal antibody. (B) Electrophoretic mobility shift assay was performed on nuclear extracts from endothelial cells transduced with the empty vector IκBmt or a combination of IκBmt and TRAF2 or TRAF6. Cells were left unstimulated (unstim), or stimulated with TNF (10 ng/mL) or LPS (100 ng/mL) for 30 minutes prior to harvesting nuclear extracts.