Figure 4.

Defective TNFα-induced IκBα phosphorylation and degradation and IκB kinase activity in the absence of NEMO/IKKγ. (A) IκBα phosphorylation. (Top) Wild-type and NEMO/IKKγ-deficient MEFs were treated with TNFα (10 ng/ml) for the indicated times followed by Western blot analysis of cytoplasmic extracts with anti-phospho-IκBα-specific antibody. (Bottom) Western blot of the same extracts with anti-IκBα antibody as a control to show IκBα protein expression in mutant cells. (B) IκBα degradation. Wild-type (top) and NEMO/IKKγ-deficient (bottom) MEFs were treated with TNFα (10 ng/ml) for the indicated times followed by Western blot analysis with anti-IκBα specific antibody. (C) IκB kinase activity. Wild-type and NEMO/IKKγ-deficient MEFs were either left in medium alone or treated with TNFα (10 ng/ml) for 7 min. Whole cell extracts were immunoprecipitated with anti-IKKα antibody and in vitro kinase activity was determined as described in Materials and Methods using GST–IκBα (1–72) (left) or GST–IκBα (1–72) S/A (right) as a substrate. One representative experiment of four is shown.