IKKβ inhibits IFN-γ signaling in macrophages. (A) Peritoneal macrophages from IKKβ^ΔMye and IKKβ^f/f mice were stimulated in vitro with 100 ng/ml LPS in combination with 100 U/ml recombinant mouse IFN-γ, and protein extracts were prepared at the indicated time points. NOS2 expression, pY-Stat1, and pY-Stat3 were measured by immunoblot analysis. (B) Peritoneal macrophages from IKKβ^ΔMye and IKKβ^f/f mice were stimulated in vitro, with IFN-γ and MHC class II expression analyzed by FACS. Representative data are shown from at least three independent experiments. (C) BMDMs were infected with recombinant adenovirus expressing a dominant-negative inhibitor of IKKβ (Ad-IKKβ^dn) or EGFP (Ad-GFP) 48 h before stimulation with LPS. NOS2 and pY-Stat1 expression was measured by immunoblot analysis at the indicated time points. (D) BMDMs were infected with Ad-IKKβ^dn or Ad-GFP before stimulation with LPS/IFN-γ, and IL-12p40 production was measured in cell culture supernatants by ELISA. Data are represented as mean ± SEM of n = 4. (E) RAW 264.7 macrophages were transfected with GAS luciferase reporter and either empty vector (pCDNA) or cDNA expressing IKKβ, IκBα, or SOCS1. After LPS/IFN-γ stimulation for 6 h, GAS reporter activity was measured by dual luciferase activity assay. Data are represented as relative light units (RLU) normalized for transfection efficiency with pRLTK.