In vitro transcription and translation of either pCS2+ Myc-MDM2 or pCS2+-FLAG-PER2, β-TrCP1MDM2, MDM2(C470A), and p53 were carried out using the SP6 high-yield TNT system (Promega) following the manufacturer’s instructions. As indicated in each case, aliquots (1 to 4 μl) of the indicated recombinant proteins were preincubated for 15 min at room temperature to allow complex formation before adding NP-40 lysis buffer. Epitope blocking was performed by preincubating in vitro the transcribed and translated FLAG-MDM2(C470A) with α-4B11, α-4B2, or α-SMP14 antibody (0.1 mg/ml each; Calbiochem) for 2 hours at 4°C before adding recombinant Myc-PER2. Binding reactions were allowed to proceed overnight at 4°C with rotation. In other experiments, binding of Myc-MDM2 or Myc-MDM2(C470A) proteins to FLAG-PER2 was evaluated in λPPase or CK1ε-treated samples as described in the section below. Reactions were diluted in NP-40 lysis buffer, and complexes were immunoprecipitated using antibodies recognizing the FLAG epitope (Sigma) and protein A beads (50% slurry) as described earlier.

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