FIG. 4.

Dexamethasone prevents TNF-α–induced alterations in the tight junction complex. Confluent BRECs were treated with 50 ng/ml Dex 18 h before TNF-α treatment (5 ng/ml, 6 h). Whole-cell extracts were assayed for (A) ZO-1, (B) claudin-5, and (C) occludin immunoreactivity by Western blotting as described in research design and methods. Representative Western blots for each tight junction protein and β-actin (loading control) are presented above each respective graph. The results are normalized to β-actin and represent the mean ± SEM of at least five independent experiments and are expressed as the relative amount compared with control. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from control; #P < 0.05, ##P < 0.01, ###P < 0.001, significantly different from TNF-α, as determined by ANOVA followed by Bonferroni post hoc test. D: Cells were immunolabeled for ZO-1, claudin-5, and occludin and 10 confocal Z-stacks were taken through 2.56 μm and projected into one image. These results are representative of four independent experiments. Scale bar, 25 μm.