Fig. 6.

Increased p53 levels do not translate to increased transcriptional activity. (A) PG13-Luc activity was assayed using H1299 cells transfected with Myc-LacZ, p53, Mdm2, and CA-Src. Twenty-four hours posttransfection, cells were treated with DMSO or 0.3 μM of the Nedd8-activating enzyme inhibitor MLN4924 for 16 h. (B) H1299 cells were treated the same as in A, except luciferase was assayed using Maspin-Luc and Myc-LacZ. (C) maspin-luciferase activity was examined in H1299 cells transfected with p53, Mdm2 Y281-302F, and CA-Src. (D) Luciferase assay of MCF7 cells transfected with either Maspin-Luc or Mutant Maspin-Luc (MT) and Myc-LacZ was conducted. Twenty-four hours after transfection, cells were treated with DMSO or 10 μM PP1 for 16 h. (A–D) Y axis measurements are relative luciferase units (RLU). The RLU were calculated from the ratio of luciferase/β-gal activity. Error bars represent SD of three separate experiments. *^,#P < 0.05. (E) MCF7 cells were treated with DMSO or 10 μM PP1 and subjected to immunoblotting for Maspin and GAPDH. (F) In response to activated Src, Mdm2 is phosphorylated and selectively binds to Ubc12 to function as a neddylating enzyme. The respective inhibitors to the pathway are denoted.