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. 2013 May 13;288(27):19450–19458. doi: 10.1074/jbc.M113.467670

FIGURE 5.

FIGURE 5.

VDR-IKKβ interaction blocks IKK complex formation and IKKβ phosphorylation. A, effects of 1,25(OH)2D3 treatment on IKK complex formation. VDR+/− MEFs were pretreated with 1,25(OH)2D3 (30 min) followed by TNFα treatment (15 min). Cell lysates were precipitated (IP) with anti-IKKγ antibodies, and the precipitates were blotted (IB) with anti-p-IKKα/β (Ser-177) or anti-IKKα/β antibodies as indicated. B, IKK enzymatic assays. VDR+/− MEFs pretreated with or without 1,25(OH)2D3 were stimulated by TNFα for 0, 5, 15, 30, and 60 min as indicated. The IKK complex was precipitated with anti-IKKγ antibodies at the indicated time points, and kinase activity to phosphorylate IκBα was measured using GST-IκBα substrate in the presence of [γ-32P]ATP. Phosphorylated GST-IκBα was visualized by autoradiography. Levels of IKKβ were analyzed by Western blotting. C, effects of 1,25(OH)2D3 on IKKβ phosphorylation. VDR+/− or VDR−/− MEFs were pretreated with 1,25(OH)2D3 for 0, 15, 30, and 60 min as indicated, followed by 15 min of TNFα stimulation. IKKβ phosphorylation was assessed by Western blotting with anti-p-IKKα/β (Ser-177) antibodies. D, effects of 1,25(OH)2D3 on IκBα degradation. VDR+/− and VDR−/− MEFs were pretreated with 1,25(OH)2D3 for 0, 5, 15, 30, and 60 min, followed by 15 min of TNFα treatment, and IκBα protein levels were assessed by Western blotting with anti-IκBα antibodies. E, HEK293 cells were transfected with empty vector or increasing amounts of HA-hVDR (0.25 or 0.5 μg). After the transfected cells were stimulated with TNFα for 15 min, cell lysates were blotted with anti-p-IKKα/β (Ser-177), anti-HA, or anti-β-actin antibodies as indicated. F, luciferase reporter assays. HEK293 cells were cotransfected with pNF-κB-Luc and empty vector control (Ctrl); IKKβ(EE), IKKβ(AA), or IKKβ (WT); and VDR as indicated. The transfected cells were treated with ethanol or 1,25(OH)2D3 for 24 h followed by luciferase activity assays. ***, p < 0.001. Note that IKKβ(EE)-induced NF-κB activity cannot be suppressed by VDR overexpression regardless of 1,25(OH)2D3 treatment.