Figure 2. Expression of IKKβ[S177E] induces the autophosphorylation of Ser¹⁸¹ and activation of IKKβ.

(A) MEFs from IKKα-deficient mice stably expressing HA-tagged wild-type IKKβ (WT), IKKβ[S177E] (S177E) or IKKβ[S177A] (S177A) or empty vector (EV) were incubated for 16 h with 1.0 μg/ml (WT and EV), 0.2 μg/ml (S177A) or 0.1 μg/ml (S177E) doxycycline to induce the expression of these proteins, and then for 1 h without (−) or with (+) 5 μM NG25 or 5 μM BI605906 and lysed. Extract [20 μg (EV, S177E and S177A) or 80 μg (WT) protein] were analysed by immunoblotting with the antibodies indicated. (B) As in (A), except that Ser¹⁸¹ phosphorylation was studied in MEFs stably expressing HA–IKKβ[D166A/S177E] and HA–IKKβ[S177E], and no inhibitors were present. (C and D) HA-tagged IKKβ[S177E] was transfected into HEK-293 cells, immunoprecipitated from the cell extracts, incubated without (−) or with (+) PP1γ and assayed for IKKβ activity (C) or immunoblotted with antibodies that recognize IKKβ phosphorylated at Ser¹⁸¹ or all forms of IKKβ (D). The results in (C) are means±S.E.M. of duplicate determinations. Similar results were obtained in two other independent experiments.