Figure 6. MDM2 interacting peptides derived from the sequence of CK1α can inhibit the E3 ubiquitin ligase function of MDM2 in vitro.

In a peptide competition ubiquitination assay, untagged p53 protein purified from Sf9 insect cells was incubated with ubiquitin, UBE1 (E1), UbcH5a (E2) and ATP in the presence of full-length MDM2 pre-incubated with selected CK1α peptide 1, 2, 28 or 35, or BOX-I/−V) MDM2 ligands. Reactions were performed for 5 min at 37°C. Samples were analysed by immunoblotting with an anti-p53 antibody. A control with no MDM2 (“-E3”, peptide 2+ p53 only) and a DMSO control (“D”) were included.