Effect of MDM2 on activation of p65 promoter in transient transfection assays.

EU-4 cells were cotransfected with 10 μg pKBCAT (containing the p65 promoter) and either increasing amounts (1, 5, and 10 μg) of wt p53 (lanes 2-4) or increasing amounts (1, 5, and 10 μg) of MDM2-sense in the presence (lanes 5-7) or absence (lanes 8-10) of a fixed amount (10 μg) of wt-p53. The pKBCAT was cotransfected with 10 μg MDM2-antisense plasmid (lane 11) as control. Total concentration of DNA was adjusted to 30 μg per transfection with empty pCMV-neo-Bam vector. Electroporation was performed at 300 V and 950 μF. At 48 hours after transfection, cell extracts were analyzed for CAT protein expression with a CAT ELISA kit. Data represent mean ± SD of 3 independent experiments. A value of 1 was assigned to CAT expression from the transfection with pKBCAT plasmid alone.