FIGURE 7.

The canonical IKKs act synergistically with IRF7 to stimulate IFNB transcription by an NFκB-independent mechanism. A, Gen2.2 cells were incubated for 1 h in the absence (no drug) or presence of 3 μm MLN4924 or 10 μm BI605906, then stimulated for 30 min with 1 μg/ml of CL097. Cell extracts were prepared and subjected to SDS-PAGE, followed by immunoblotting (IB) with the indicated antibodies. Similar results were obtained in two other independent experiments. B, same as A, except that the cells were stimulated for 1 h with 1 μg/ml of CL097 and the levels of IFNB and IKBA mRNA were measured by quantitative PCR. The results are plotted as the fold-increase in mRNA relative to the level determined in unstimulated cells. The experiment was performed in 24-well plates with two wells being used for each concentration of inhibitor studied and the results being averaged. Similar results were obtained in two other independent experiments. C, HEK293 cells were transfected with different combinations of plasmids encoding HA-tagged forms of IRF7, IKKα, IKKβ, or a catalytically inactive mutant of IKKβ. 48 h after transfection, cell extracts were prepared and subjected to SDS-PAGE, followed by immunoblotting with an anti-HA antibody. Similar results were obtained in several other independent experiments. D, lysates of HEK293 cells transfected with the indicated plasmids were subjected to HA immunoprecipitation and treated with phage λgt10-phosphatase in the absence or presence of phosphatase inhibitors. Samples were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with an anti-HA antibody. Similar results were obtained in a second independent experiment. E, HEK293 cells were transfected with a reporter plasmid encoding a firefly luciferase gene under control of the IFNβ promoter, together with the indicated concentrations of plasmid DNA encoding HA-tagged forms of IKKα and IKKβ. 48 h after transfection, cell lysates were obtained and used to quantify the relative amounts of luciferase or subjected to SDS-PAGE followed by immunoblotting with anti-HA. Transfections and luciferase measurements were performed in triplicate and the results are presented as mean ± S.D. Similar results were obtained in a second independent experiment. As in E, except that different combinations of the indicated plasmids were transfected into HEK293 cells together with a luciferase IFNβ reporter and relative luciferase measurements were performed 48 h later.