Deletion and mutation analysis of the p65 promoter activity.

(A) Schematic representation of p65 promoter-CAT reporter plasmids as described previously29: pKBCAT containing the full-length promoter; p65F1CAT, p65F3CAT, and p65F5 CAT containing a series of deleted promoters; p65F6CAT containing deleted promoter with the third Sp1-binding site mutated by site-directed mutagenesis; P65F1xxCAT, the p65F1CAT in which the first 2 Sp1 sites were mutated by site-directed mutagenesis. (B) Transient transfection and CAT assay. EU-4 cells were cotransfected with 10 μg MDM2 sense or antisense plasmid plus 10 μg p65 promoter constructs. Electroporation and CAT assays were performed as described in Figure 4. Fold CAT induction shows the comparison of cotransfection of MDM2 plasmid and promoter-CAT construct with transfection of individual promoter-CAT construct alone.