Fig. 8.

(a) Western blot analysis of TLR4 expression in HeLa hVEC. Cells were seeded at a density of 10⁶/well in 24-well plates and treated with LPS (10 μg/ml for 6 h), or LPS-induced (10 μg/ml for 6 h) cells were treated with RVFHbαP (70.45 μM for 1 h) or scrambled peptides (70.45 μM for 1 h). At the end of treatment, cells were collected and lysates were prepared and analyzed for TLR4 and β-actin by Western blotting as detailed in Materials and Methods. Expression of TLR4 was upregulated in LPS-induced cells and significantly blocked following the treatment of LPS-induced cells with RVFHbαP in respect to the scrambled peptide. Values are calculated as the means ± SD of triplicate determinations and are representative of at least three separate experiments performed on different days. Similar inhibition of TLR4 was observed when hVECs were treated with anti-TLR4 antibody before being induced with LPS. A representative image of Western blot analysis of TLR4 expression is shown. β-Actin blotting confirmed roughly equivalent loading of protein samples. (b) A quantitative assessment of the intensity of each band was determined by densitometry. (c) Protein ELISA was carried out to confirm the data obtained by Western blot analysis, and the results are in agreement with Western blotting results. Levels of significance (**, P < 0.001 compared with the LPS- and LPS-nRVFHbαP-treated groups) were calculated by an ANOVA test followed by a Bonferroni analysis.