FIGURE 9.

Effect of iberin on NF-κB signaling pathway. A, immunoblot analysis of nuclear translocation of p65. The RAW 264.7 cells were pretreated with iberin for 30 min and then treated with LPS (100 ng/ml) for 4 h. After incubation, the nuclear fractions were prepared, and immunoblotting was performed with the anti-p65 and anti-lamin A antibodies. A representative immunoblot (top) and densitometric analysis of p65 protein normalized to lamin A (bottom) are shown. Error bars represent S.D. B, immunoblot analysis of IκB degradation. The cells were pretreated with iberin for 30 min and then treated with LPS (100 ng/ml) for 30 min. After incubation, immunoblotting was performed with the anti-IκB and anti-GAPDH antibodies. A representative immunoblot (top) and densitometric analysis of IκB protein normalized to GAPDH (bottom) are shown. Error bars represent S.D. C and D, immunoblot analysis of phosphorylation of mitogen-activated protein kinases. The RAW264.7 cells were pretreated with iberin for 30 min and then treated with LPS (100 ng/ml) for 30 min. A representative immunoblot (C) and densitometric analysis of phosphorylated (p-) MAPKs normalized to total MAPKs (D) are shown. The values of LPS-treated cells are shown as a control (100%). Error bars represent S.D. E, effect of quercetin and Q4′G on NF-κB activation mediated by MyD88. The HEK293 cells were transiently co-transfected with the expression plasmid for the adaptor protein MyD88 or empty vector (Mock) and NF-κB reporter plasmid. After transfection, the cells were treated with quercetin (QUE) or Q4′G for 24 h. #, p < 0.01 versus Mock; ***, p < 0.005 and **, p < 0.01 versus vehicle (MyD88 expression). Error bars represent S.D. F, effect of quercetin and Q4′G on poly(I:C)-induced phosphorylation of TBK1 (p-TBK1). RAW264.7 cells were pretreated with quercetin or Q4′G (10 μm) for 30 min and then treated with poly(I:C) (10 μg/ml) for 30 min.