T-bet enhancer interacts with IL-12–activated Stat4 and transactivates reporter gene expression. (A). EMSA in NK92 cells. Nuclear extracts were prepared from NK92 cells treated with IL-12 for 15 minutes. A indicates site A within the identified distal T-bet enhancer; mA, mutated site A. Competition was performed with 10-, 100-, or 1000-fold unlabeled probe. Equal amount of specific anti-Stat1 (S1) and anti-Stat4 Abs (S4) was used for neutralization. (B) EMSA in CD8 T cells. Nuclear extracts were prepared from cells 24 hours after stimulation. Site A indicates the STAT-binding site within the identified distal T-bet enhancer; site C, a potential STAT-binding site located 940-bp upstream of the transcription initiation site. Equal amount of specific anti-Stat1 (S1) and anti-Stat4 (S4) Abs was used for neutralization. (C) T-bet enhancer constructs containing STAT-binding motifs respond to IFNγ/Stat1 by transactivating Luciferase reporter expression. Total of 5 constructs were made. No. 1 contains 5′ nearby region; no. 2 contains 3′ nearby region; no. 3 is the exact conserved region; no. 4 is the minimal conserved region containing 2 potential STAT-binding motifs; and no. 5 is the conserved region without STAT-binding motifs. HeLa cells were transfected with the indicated constructs and treated with human IFNγ for 24 hours, and luciferase activities were measured. (D) Point mutations were made, and reporter assays were performed. (E) JAK2-mediated Stat4 activation induces reporter gene expression. U3A cells were cotransfected with the conserved T-bet enhancer region (construct no. 3, 100 ng), along with JAK2 (100 ng), in combination with mouse or human Stat4 (500 ng) plasmids. (F) T-bet enhancer constructs containing STAT-binding motifs respond to both Stat1 and Stat4. The indicated T-bet enhancer reporter constructs were cotransfected with human Stat1 or Stat4, along with JAK2 into EL-4 cells. (C-F) Data shown are the means plus or minus SD from 3 independent experiments.