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. 2010 Jun 23;285(35):26987–26997. doi: 10.1074/jbc.M110.121061

FIGURE 5.

FIGURE 5.

Crotepoxide inhibits TNF-induced IκBα degradation, IκBα phosphorylation, and IKK activation. Crotepoxide inhibited TNF-induced NF-κB activation and IκBα degradation. KBM-5 cells were incubated with crotepoxide (50 μm) for 2 h and then treated with TNF (0.1 nm) for the indicated times. A, nuclear extracts were analyzed for NF-κB activation by EMSA. B, cytoplasmic extracts were analyzed for IκBα degradation by Western blotting with antibodies against anti-phospho-IκBα and anti-IκBα. Equal protein loading was evaluated by β-actin. C, shown is the effect of crotepoxide on TNF-induced IκBα phosphorylation. Cells were preincubated with crotepoxide (50 μm) for 2 h, incubated with N-acetyl-leucyl-leucyl-norleucinal (ALLN; 50 μg/ml) for 30 min, and then treated with TNF (0.1 nm) for 10 min. Cytoplasmic extracts were fractionated and then subjected to Western blotting with phospho-specific IκBα antibody. The same membrane was reblotted with β-actin. Ub, ubiquitin. D, crotepoxide inhibited TNF-induced IKK activation. KBM-5 cells were preincubated with crotepoxide (50 μm) for 2 h and then treated with TNF (1 nm) for the indicated times. Whole-cell extracts were immunoprecipitated with antibody against IKK-α and analyzed by an immune complex kinase assay. To examine the effect of crotepoxide on the level of expression of IKK proteins, we fractionated whole-cell extracts on sodium dodecyl sulfate-polyacrylamide electrophoresis gels and examined by Western blot analysis with anti-IKK-α and anti-IKK-β antibodies. E, crotepoxide directly affected TNF-induced IKK activation. Whole-cell extracts (WCE) were prepared from KBM-5 cells treated with TNF (1 nm) and immunoprecipitated with anti-IKKα antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentrations of crotepoxide. F, crotepoxide inhibited the phosphorylation of IKKα/β. TNF (1 nm) was exposed for an indicated time period in 2 h before crotepoxide (50 μm)-pretreated KBM-5 cells. Whole-cell extracts were prepared and then subjected to Western blotting with phospho-specific anti-IKKα/β antibody. The same membrane was reblotted with anti-IKKα and anti-IKKβ antibodies.