FIGURE 7.

Activation and phosphorylation of TBK1 and IKKε by IL-1α and TNFα. A and B, kinetics of activation of TBK1 and IKKε. MEFs were serum-starved overnight and stimulated for the indicated times using 10 ng/ml IL-1α (A) or TNFα (B). Catalytic activity was measured by immunoprecipitating TBK1 and incubating the immune complexes with GST-IRF3 in the presence of Mg[γ-³²P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were detected by autoradiography (top panel). Phosphorylation of Ser-172 on TBK1 was monitored by immunoprecipitating the phosphorylated TBK1 and IKKε with the anti-IKKε Ser(P)-172 antibody (S051C) and immunoblotting with an antibody recognizing all forms of TBK1 and IKKε (middle panel). C and D, effect of BX795 on the activation of TBK1 and IKKε. MEFs were incubated for 1 h without (–) or with (+)1μm BX795 before stimulation for 10 min with 10 ng/ml IL-1α (C) or TNFα (D). Catalytic activity and phosphorylation of TBK1 and IKKε were measured as in A and B.