FIG. 9.
Both κB and Sp1 are required for full TNF-α- or PMA-induced IκBα promoter activity. (A) Jurkat cells were transfected with 1 μg of luciferase reporter plasmid containing wild-type (0.4SK), mutant κB1 (ΔκB or mutκB1), mutant Sp1 (mutSp1), or mutant κB1/Sp1 (mutκB1/Sp1) IκBα promoter. Twenty four hours after transfection, cells were treated with TNF-α (10 ng/ml) or PMA (50 ng/ml) or left untreated for an additional 16 h. Transfection efficiency was normalized to that of Renilla luciferase (see Materials and Methods). The experiments were performed in triplicate, and the average fold induction was calculated. (B) EMSA was performed with different radiolabeled oligonucleotide probes (κB1/Sp1, mutated κB1/Sp1 [mutκB1], and κB1/mutated Sp1 [mutSp1]), using uninduced or PMA (50 ng/ml for 2 h)-induced Jurkat nuclear extract. (C) Luciferase assays were performed as described for panel A, in using 1 μg of reporter plasmids containing an 8-nt deletion (Δ8), 5-nt addition (+5), and 9-nt addition (+9) between κB1 and Sp1 sites of the IκBα promoter. Transfection efficiency was normalized to that of Renilla luciferase (see Materials and Methods). The experiments were performed in triplicate, and the average fold induction was calculated.