FIG. 5.

Effect of NF-κB inhibition on TNF-α–induced cell permeability. A: Cells were incubated with 1 μmol/l IKK VII, 30 min before the addition of 5 ng/ml TNF-α for 5 min. Whole-cell lysates were assayed for phophorylated IκBα (Ser32/Ser36) and total IκBα immunoreactivity by Western blotting. B: Cells were grown to confluence on transwell filters and then treated with 1 μmol/l IKK VII 30 min before TNF-α addition (5 ng/ml, 6 h). The monolayer permeability to 70 kDa dextran was measured as described. C–E: Adenovirus-mediated overexpression of IκBα. BRECs were transduced with AdEmpty or AdIκBα as described. C: Whole lysates of BRECs were used to detect IκBα, GFP, and β-actin (loading control) by Western blotting. D: After 28 h of adenovirus transduction, cells were exposed to 5 ng/ml TNF-α for 2 h. Total RNA was isolated, and the transcript levels of IL-8 were analyzed by qPCR. E: Cells were grown to confluence on transwell filters and after 24 h of adenovirus transduction cells were treated with 5 ng/ml TNF-α for 6 h. The monolayer permeability to 70 kDa dextran was measured. The results represent the mean ± SEM of at least three independent experiments and are expressed relative to control (Ctrl). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different as determined by ANOVA followed by Bonferroni post hoc test.