WT MEFs were transfected with the two pcDNA3.1 plasmids containing mouse Trp53 and the indicated mouse Mdm2 constructs, respectively, as well as the mCherry plasmid (pmCherry-C1), using GeneJuice (Novagen) following the manufacturer's recommendations. Briefly, a mixture of DNA plasmids (in a 1:1:1 ratio) and GeneJuice in OptiMEM was added dropwise to cell culture plates that had been seeded the day before. Reagent volumes, cell numbers, and DNA content were scaled according to plate size within the manufacture's recommended ranges. Cells were incubated for 24 h with the transfection mixture to allow for plasmid expression and then the degradation of p53 relative to mCherry was assessed through Western blot analysis.

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