Ectopic expression of FBXO31, but not FBXO31∆F, substantially reduces the half-life of MDM2. (A) qRT-PCR analysis monitoring MDM2 mRNA levels in MCF7 cells expressing vector or FBXO31 (Left) or expressing NS or FBXO31 shRNA (Right). Error bars indicate SD. (B) Cycloheximide-chase/immunoblot assay monitoring Flag-MDM2 stability in cycloheximide-treated MCF7 cells expressing GFP (control), FBXO31, or FBXO31∆F. PCNA was monitored as a control. (C, Upper) Cycloheximide-chase/immunoblot assay monitoring Flag-MDM2 stability in 293T cells expressing GFP (control), FBXO31, or FBXO31∆F at the indicated time points following treatment with cycloheximide. (Lower) Quantification. (D) Cycloheximide-chase/immunoblot assay monitoring GFP-p53 and Flag-MDM2 stability in cycloheximide-treated Mdm2−/−, p53−/− MEFs coexpressing p53, MDM2, and FBXO31 in the indicated combinations.