Figure 1.

Inhibition of Sp1 activity by MDM2. (A) Transcriptional activity of endogenous Sp1 in cells with amplified MDM2. NIH 3T3 cells and a hybrid with amplified MDM2 (MDM2+) were transfected with the synthetic Sp1-dependent promoter construct (Sp1)₃BCAT (20) and increasing amounts of an Sp1-dependent expression vector cytomegalovirus (CMV)-Sp1. RSVCAT was used to control for transfection efficiency between cell lines. CAT activity is expressed as cpm above background. Values represent the average of duplicate dishes, with <20% difference between duplicate dishes. (B) Expression of endogenous Sp1 is not affected by MDM2 amplification. Western blot analysis, using an antibody against Sp1, on cell extracts from NIH 3T3 cells and a hybrid with amplified MDM2 (MDM2+). (C) Inhibition of Sp1 activity by MDM2 and MDMX. NIH 3T3 cells were transfected with the Sp1-dependent reporter (Sp1)₃BCAT, HSVTKCAT (TKCAT), or GALCAT. Cells were cotransfected with either an Sp1 expression vector or an GAL-N expression vector in the presence of increasing amounts of an MDM2 or MDMX expression vector. CAT activity was measured as in A above and is expressed as percentage of activated levels.