FIGURE 6.

Crotepoxide inhibits TAK1 activation and nuclear translocation of p65. A, crotepoxide inhibited TNF-induced TAK1 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 TAK1 and analyzed by an immune complex kinase assay. To examine the effect of crotepoxide on the level of expression of TAK1 proteins, Western blot analysis of whole-cell extracts (WCE) was performed with anti-TAK1 antibody. B, crotepoxide directly affected TNF-induced TAK1 activation. Whole cell extracts were prepared from KBM-5 cells treated with TNF (1 nm) and immunoprecipitated with anti-TAK1 antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentrations of crotepoxide. C, crotepoxide inhibited TNF-induced p65 phosphorylation. KBM-5 cells were left untreated or pretreated with crotepoxide (50 μm) for 2 h at 37 °C and then treated with TNF (0.1 nm) for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting with antibodies against p65 and phospho-specific p65. For loading control of nuclear protein, the membrane was blotted with anti-poly(ADP-ribose) polymerase antibody (PARP). D, crotepoxide inhibited the nuclear translocation of p65. KBM-5 cells were first treated with crotepoxide (50 μm) for 2 h at 37 °C and then exposed to TNF (0.1 nm) for 15 min. Cells were centrifuged and underwent immunocytochemical analysis.