FIG. 6.

LJ inhibits both LPS-induced phosphorylation and degradation of IκB-α (A, B) as well as activation of NF-κB in BV-2 microglial cells. Cells were pretreated with the indicated concentrations of LJ for 30 min and then exposed to 100 ng/mL of LPS for 1 h. The expression levels of IκB-α, STAT1/3, NF-κB p65, β-actin, and lamin B1 were measured by western blot analysis (C, D). Densitometric results are presented as mean±SEM (n=3). NF-κB p65 was probed by anti-NF-κB p65 antibody and Texas red^®-conjugated secondary antibody. Nuclei were stained with Hoechst 33258 and representative pictures were taken with a fluorescence microscope (E, 100× magnification). Images are representative of three experiments. Scale bar: 50 μm. Cells were transiently transfected with an NF-κB reporter plasmid construct and then pretreated with the indicated concentrations of LJ for 30 min, after which they were stimulated with 100 ng/mL of LPS for 1 h. Equal amounts of cell extracts were assayed for dual-luciferase activity (F). Data are presented as the mean±SEM (n=3). Cells were pretreated with the indicated concentrations of LJ for 30 min before being stimulated with 100 ng/mL of LPS for 1 h. Nuclear extracts were tested for specific DNA binding of NF-κB by EMSA (G). **P<.01 and ***P<.001 compared with the control group. ^#P<.05, ^##P<.01, and ^###P<.001 compared with the LPS-treated group. EMSA, electrophoretic mobility shift assay; NF-κB, nuclear factor-κB. Color images available online at www.liebertpub.com/jmf