Fig. 6. Effects of EUE on LPS-induced phosphorylation of IκBα (A), degradation of IκBα (B), and activation of NF-κB in BV-2 microglial cells. Cells were pretreated with the indicated concentrations of EUE for 30 min and then stimulated with 100 ng/ml LPS for 1 h. The levels of IκBα, NF-κB p65, β-actin, and lamin B1 were measured by Western blot analysis (A?D). Densitometric results are presented as means ± S.E.M. (n=3). (E) Immunofluorescence analysis of NF-κB p65. Cells were immunostained with anti-NF-κB p65 antibodies and Texas Red?-conjugated secondary antibodies. Nuclei were counterstained with Hoechst 33258, and representative pictures were taken with a fluorescence microscope (100× magnification). Images shown are representative of three independent experiments. Scale bar: 200 μm. (F) Cells were transiently transfected with an NF-κB reporter construct, pretreated with the indicated concentrations of EUE for 30 min, and then stimulated with 100 ng/ml LPS for 1 h. Equal protein amounts of cell extracts were assayed for dual-luciferase activity. Data are presented as means ± S.E.M. (n=3). (G) Cells were pretreated with the indicated concentrations of EUE for 30 min and then stimulated with 100 ng/ml LPS for 1 h. The DNA binding of NF-κB in nuclear extracts was then assayed by EMSAs. ***p<0.001 compared with the control group. #p<0.05; ##p<0.01; and ###p<0.001 compared with the LPStreated group.
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