FIGURE 6.

BX795 increases the phosphorylation of Ser-172 and the catalytic activity of TBK1 and IKKε in response to LPS and poly(I:C). A, RAW264.7 cells were treated for 1 h without (–) or with (+)1 μm BX795 before stimulation for 45 min without (–) or with (+) 100 ng/ml LPS. TBK1 was immunoprecipitated from 1 mg of cell extract protein, and its catalytic activity was measured by incubation for 30 min at 30 °C with GST-IRF3 and Mg[γ-³²P]ATP. Reactions were terminated in SDS, proteins were resolved by SDS-PAGE, and the gel was autoradiographed (top panel). The gel was subjected to phosphorimaging analysis, and the catalytic activity of TBK1 was quantitated and normalized to that observed in the unstimulated cells (mean ± S.E., n = 3; *, p < 0.05). B, an aliquot of the cell extract in A was used to analyze the phosphorylation of TBK1 and IKKε at Ser-172 as in Fig. 3C (top panel). As a loading control, cell extract (40 μg of protein) was also immunoblotted with antibodies recognizing all forms of TBK1 and IKKε (lower panel). This antibody also recognizes a nonspecific band migrating slower than TBK1 and IKKε. C, RAW264.7 cells were incubated for 1 h with the indicated concentrations of BX795 and then either left unstimulated (–) or stimulated (+) for 45 min with 100 ng/ml LPS. The phosphorylation of TBK1 and IKKε at Ser-172 was then examined by immunoprecipitation/immunoblotting as in Fig. 3C. D, bone marrow-derived macrophages were incubated for 1 h without (–) or with (+)1 μm BX795 followed by stimulation for 30 or 60 min with 100 ng/ml LPS. Cell extract (20 μg protein) was then immunoblotted with the antibodies indicated. E, the experiment was performed as in D, but the cells were stimulated for 60 min with 10 μg/ml poly(I:C). F, RAW264.7 cells were stimulated for 45 min with 100 ng/ml LPS, TBK1 was immunoprecipitated from 0.5 mg of cell extract protein, and its ability to phosphorylate GST-IRF3 (2 μm) was measured at varying concentrations of BX795 using Mg[γ-³²P]ATP. Reactions were terminated in SDS, proteins were resolved by SDS-PAGE, and the gel was autoradiographed (top panel). The gel was subjected to phosphorimaging analysis, and the catalytic activity of TBK1 was quantitated and normalized to that measured in the absence of inhibitor (mean ± S.E., n = 4).