Functional elements required for IL12RB1 transcriptional activation. (A) Schematic presentation of the IL12RB1 promoter deletion constructs. A series of 5′ promoter deletion constructs were cloned into pGL3-basic vector and were stably integrated into RAW264.7 cells. The number of each construct corresponds to its 5′ end. (B) RAW264.7 cells stably integrated with IL12RB1 deletion constructs were cultured without serum at 1 × 10⁵ cells/plate for 24 hours. Cells were left untreated or were treated with one of the following: 10 ng/mL IFN-γ, 10 ng/mL IL-15, or 1 μg/mL LPS for 8 hours before lysate preparation for luciferase assays. Luciferase activity from 2 independent clones of each deletion construct was examined. Basal luciferase activity of untreated cells was generally similar among all the examined clones. Results are expressed as fold induction over untreated controls (mean ± SD). (C) The nucleotide sequence of the region required for the IL12RB1 promoter activation by IFN-γ and IL-15, potential binding sites for transcription factors, are indicated by bold capital letters. Arrows identify the 5′ end of deletion constructs, and nucleotides used as primers for ChIP assays were underlined. (D) Schematic presentation of the second series of IL12RB1 promoter deletion mutant. Black boxes represent binding elements for indicated transcription factors. (E) RAW264.7 cells stably integrated with a second series of promoter deletion mutant (as in panel D) were cultured and plated as described in panel B. Cells were left untreated or were treated with 10 ng/mL IFN-γ or 10 ng/mL IL-15 for 8 hours before lysate preparation and luciferase assays. Basal luciferase activity of untreated cells was generally similar among the examined clones. Fold inductions by IFN-γ or IL-15 are expressed as described in panel B.