IL-15, synergistically with IFN-γ, activates IL12RB1 gene transcription. (A) RAW264.7 cells were left untreated (unstim) or were stimulated with either of the following: 1 μg/mL LPS, 10 ng/mL IL-15, 10 ng/mL IFN-γ, or combination IL-15 and IFN-γ for 4 hours. In some experiments, cells were pretreated with 100 mM TSA before stimulation. Total RNA was prepared for Northern blot analysis. Gene expression of IL12RB1 and a picture of the ethidium bromide (EtBr)–stained gel are shown. (B) RAW264.7 cells stably integrated with pGL3-2508 luciferase construct were prepared as described for Figure 2B. Cells were left untreated or were treated with 10 ng/mL IFN-γ, 10 ng/mL IL-15, combination IFN-γ and IL-15 for 6 hours with or without the addition of 100 mM TSA before lysate preparation and luciferase assays. Basal luciferase activity of untreated cells was generally similar among all the examined clones. Fold inductions by IFN-γ or IL-15 are expressed as described for Figure 2B. (C) IL-15 and IFN-γ synergistically enhanced protein binding to IRE/ISRE. RAW264.7 cells were left untreated or were treated with IFN-γ, IL-15, or combination IFN-γ and IL-15 for 30 or 60 minutes, and nuclear extracts were prepared for EMSA. EMSA was carried out as described in Figure 3A; inducible protein binding is shown. (D) Acetylation of histone H3 at the IL12RB1 gene is induced by IFN-γ and IL-15. RAW264.7 cells were left untreated (unstim) or were treated with 10 ng/mL IFN-γ or 10 ng/mL IL-15 for the indicated time. After treatment, chromatin was extracted and immunoprecipitated with anti–acetyl-histone H3 or anti–acetyl-histone H4. PCR analyses of DNA products from immunoprecipitation were carried out as described in “Materials and methods.” (E) Mouse peritoneal macrophages were prepared as previously described,³⁰  and the ChIP assays were carried out as described for Figure 4D. Acetylation levels of histone H3 at the IL12RB1 gene are shown.