FIG. 2.

Identification of inducer-specific requirements of activator binding sites for the TNF-α promoter activity in response to ionophore and virus. (A) Sequence of the TNF-α promoter from −200 to −20 nt. Activator binding sites are boxed and colored, and sites that are capable of binding two activators contain two colors. The mutations (M's) tested in panel B are shown below the wild-type sequence. The TATA box is shown in a black box. (B) Relative activities of TNF-α-Luc fusion constructs containing mutations (M's) in activator binding sites in ionophore- and virus-stimulated 68-41 T cells. 68-41 T cells were transfected with 1 μg of the wild-type −200 TNF-α promoter-Luc reporter (WT) or with isogenic reporters containing the mutations in different activator binding sites as shown in panel A. A Renilla luciferase control plasmid (1 μg) was cotransfected in all cases. Cells were then stimulated with ionomycin or Sendai virus as described in Materials and Methods, and luciferase assays were performed and normalized to Renilla luciferase activity. The histograms show the results of four independent experiments. Error bars represent the standard errors of the mean.