FIG. 2.

TNF-α alters tight junction proteins content and cell localization. BRECs were treated with 5 ng/ml TNF-α for 0.5 and 6 h. Whole-cell extracts were assayed for ZO-1 (A), claudin-5 (B), and occludin (C) immunoreactivity by Western blotting. 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 (Ctrl). *P < 0.05, **P < 0.01, significantly different from control as determined by ANOVA followed by Dunnett post hoc test. Total RNA was isolated after 6 h of TNF-α treatment, and the transcript levels of ZO-1 (D), claudin-5 (E), and occludin (F) were analyzed by qPCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Results represent the mean ± SEM of eight independent experiments and are expressed as the relative amount compared with control conditions. *P < 0.05, ***P < 0.001, significantly different from control as determined by Student t test. G: Cells were immunolabeled for ZO-1, claudin-5, and occludin 6 h after TNF-α treatment, and 10 confocal Z-stacks were taken through 2.56 μm and projected into one image. Arrows indicate continuous staining at cell borders. These results are representative of four independent experiments. Scale bar, 25 μm.