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Fig. 3. I{kappa}B{alpha}(Y42F) inhibits NF{kappa}B activation following pervanadate and H/R treatment. (A) HeLa cells were co-infected with Ad.I{kappa}B{alpha}(Y42F) or Ad.BglII together with Ad.NF{kappa}BLuc 24 hours before treatment. Cells were treated with UV (50 J/m2), TNF-{alpha}(10 ng/ml), pervanadate (50 µM) and H/R (5 hours hypoxia, 6 hours reoxygenation) then harvested 6 hours after treatment. Whole cell extracts were normalized by total protein content and subjected to luciferase assay. NF{kappa}B activity was determined by the relative luciferase activity (as light units). The relative NF{kappa}B activation was calculated by deducting the mean NF{kappa}B baseline activation for each vector group (in the absence of stimulation) from the corresponding individual values from the same vector group in the presence of stimulus. The relative NF{kappa}B activation is plotted for each individual stimulus (±s.e.m., n=6). (B) Percent NF{kappa}B inhibition by I{kappa}B{alpha}(Y42F) for each experimental point was calculated using the following formula: percent inhibition=1—(relative activation of each Ad.I{kappa}B{alpha}(Y42F) infected sample/mean relative activation of the Ad.BglII infected group). Results depict the mean (±s.e.m.) for n=6 independent data points in each group.