Effects of CBP expression on IL12RB1 promoter activity and IRF3 phosphorylation. (A) RAW264.7 cells were stably integrated with pGL3-3875 of IL12RB1 luciferase construct, together with CBP-HA expression plasmid. Total cellular lysates were prepared from stably integrated clones, and the expression levels of CBP were analyzed by Western blotting using anti-HA mAb. (B) Overexpression of a coactivator protein CBP increases IFN-γ–mediated IL12RB1 transcription. The stably integrated clones shown in panel A were cultured and plated as described for Figure 2B. Cells were left untreated or were treated with either of the following: 10 ng/mL IFN-γ (□), 10 ng/mL IL-15 (▦), or 1 μg/mL LPS (not shown) for 8 hours before lysate preparation and luciferase assays. Basal luciferase activity of untreated cells was generally similar among all the examined clones. Fold inductions are shown. (C) Neither IFN-γ nor IL-15 induces gene expression of IRF3. RAW264.7 cells were left untreated (unstim) or were treated for 4 hours with 1 μg/mL LPS, 10 ng/mL IL-15, or 10 ng/mL IFN-γ. Total RNA was prepared for Northern blot analysis using IRF1, IRF2, IRF3, and IRF7 cDNA probes. Gene expression of the indicated gene and a picture of ethidium bromide (EtBr) are shown. (D) RAW264.7 cells were treated as described for Figure 5A. Total cellular lysates were prepared and immunoprecipitated with anti-IRF3 pAb. The phosphorylation of IRF3 was detected by Western blot analysis using antiphosphoserine mAb. As a control, 10% of each lysate was used to detect IRF3 by anti-IRF3 pAb.