Fig. 4.

MDM2 and MDMX are required for maintaining levels of p73 and E2F1 in cells. (A) H1299 cells were transfected with siMDM2, siMDMX, or control siRNA for 48 h and levels of the indicated proteins were determined by immunoblotting. (B) H1299 cells were treated with DMSO or MEL23 (15 μM) for 24 h and levels of the indicated proteins were determined by immunoblotting. (C) H1299 cells were treated with increasing concentrations of SP-141 as indicated. Levels of MDM2, MDMX, TAp73α, and E2F1 were measured by immunoblotting. (D) H1299 cells were treated with siMDM2 or control siRNA and p73 mRNA was quantified by qRT-PCR. **P < 0.01 (one-way ANOVA). (E) H1299 cells were transfected with constructs expressing Myc-MDM2 (2 μg), HA-E2F1 (1.7 μg), and/or HA-TAp73 (0.3 μg), as indicated. Twenty-four hours later, cells were treated with MG132 (20 μM) for 4 h, in the absence or presence of Nutlin-3 (40 μM), as indicated. Cell lysates were prepared and subjected to immunoprecipitation with anti-Myc antibody to pull down MDM2, followed by immunoblotting with anti-HA antibody to detect E2F1 and TAp73. (F) The ratios of MDM2:E2F1 or MDM2:p73 (performed either with Myc-MDM2 or untagged MDM2) were quantified by densitometry and graphed as averages of six independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.00001. All data represent at least three biological replicates (error bars represent SEM).