GA inhibits TNF-induced IκBα degradation, IκBα phosphorylation, and IKK activation. (A) GA inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 5 μM GA for 4 hours, treated with 0.1 nM TNF for the indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of GA on TNF-induced degradation of IκBα. KBM-5 cells were incubated with 5 μM GA for 4 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared and analyzed by Western blotting with antibodies against anti-IκBα. Equal protein loading was evaluated by β-actin. (C) Effect of GA on the phosphorylation by IκBα by TNF. Cells were preincubated with 5 μM GA for 4 hours, incubated with 50 μg/mL ALLN for 30 minutes, and then treated with 0.1 nM TNF for 10 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blotting with phospho-specific IκBα antibody. The same membrane was reblotted with β-actin. (D) Effect of GA on the activation of IKK by TNF. KBM-5 cells were preincubated with 5 μM GA for 4 hours and then treated with 1 nM TNF 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 GA on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis with anti–IKK-α and anti–IKK-β antibodies.