Stable expression of transfected (ectopic) MDM2 and its effect on the expression of p65, Sp1, and NFκB-regulated gene products cIAP1, cIAP2, and XIAP.

MDM2 mRNA in EU-4 cells was detected by Northern blot assay (A), and the protein levels of MDM2, p65, Sp1, cIAP1, cIAP2, and XIAP in MDM2-transfected and control cells were detected by Western blot assay (B). Total RNA and protein were extracted simultaneously from parental EU-4 cells (lane 1) and EU-4 cells stably transfected with either pCMV plasmid-neo alone (lane 2) or pCMV-MDM2 (lanes 3-6). Stably transfected cells were derived from G418-resistant single clones maintained in culture for 3 months. Northern blot and Western blot analyses were performed as described in “Materials and methods.”