FIGURE 3.

KLHL21 negatively regulates the activation of NF-κB signaling. 293T cells transfected with KLHL21-3F were treated with 20 ng/ml TNFα. Nuclear translocation of p65 (A), total IκBα protein (B), and phosphorylated IκBα (Ser-32) (E) at the indicated time points was analyzed by Western blotting. 293T cells transfected with pcDNA3 empty vector were used as a control. C, 293T cells were transfected with pooled siRNA specifically targeting human KLHL21 at the indicated concentrations. The level of KLHL21 mRNA was analyzed at 24 h after the transfection (n = 3). D and F, 293T cells were transfected with 25 nm negative control siRNA and siKLHL21 as indicated. The degradation and reaccumulation of IκBα proteins (D) and phosphorylated IκBα protein (F), respectively, in response to 20 ng/ml TNFα treatment were analyzed by Western blotting. The density ratio of p65/Lamin B (A) quantified by densitometric scanning is shown on the right (n = 3). Data from A and C are plotted as means ± S.E. (error bars). All experiments were performed at least three times. WCL, whole cell lysates; IB, immunoblotting; *, p < 0.05; **, p < 0.01.