Figure 3. IKKβ phosphorylated at Ser¹⁷⁷ has little activity if Ser¹⁸¹ is not phosphorylated.

(A) MEFs from IKKα-deficient mice were incubated for 1 h without (−) or with (+) 5.0 μM BI 605906 or 2 μM NG25, then stimulated for 10 min with 5.0 ng/ml IL-1. The endogenous IKKβ was immunoprecipitated from 0.2 mg of cell extract protein and assayed for activity. (B) The immunoprecipitates from (A) were denatured in SDS before and after the assay of IKKβ, and aliquots of each sample were subjected to SDS/PAGE, transferred on to PVDF membranes and immunoblotted with antibodies that recognize IKKβ phosphorylated at Ser¹⁷⁷ or Ser¹⁸¹ or all forms of IKKβ.