Figure 4. MDM2 binds to a dominant small motif within CK1α.

(A) Biotinylated CK1α peptides were used to generate streptavidin-based peptide aptamer affinity columns for cell lysate MDM2 interaction assessment (B), or were coupled to streptavidin coated plates for direct ELISA binding assay (C). (B) Pre-cleared A375 lysate was applied to peptide aptamer affinity columns. A DMSO control was included. Eluates from the peptide aptamer affinity columns were analyzed on 10% gels and MDM2 binding was analyzed on immunoblots probed with anti-MDM2. Quantification of bound MDM2 protein level was performed using Scion Image software. (C) Mapping of MDM2 binding to overlapping CK1α peptides by ELISA. Overlapping CK1α peptides were coupled to streptavidin coated plates. Purified GST-cleaved MDM2 was added to each peptide and antibody against MDM2 and the appropriate secondary antibody were used to quantify the binding using ECL (in relative light units (RLU)). A DMSO control was included and values were normalised against the BSA background. The data are representative of three independent experiments in which each data point was assessed in triplicate.