Figure 3. Simulation of HEY1 phosphorylation at residue Ser-68 specifically alters HEY1 protein–protein interactions.

(A) Whole-cell extracts from U2OS cells previously transfected with expression vectors for Flag-tagged HES1 or HEY2 were incubated with GST fusion proteins of HEY1 or HEY1-S68D coupled with Sepharose beads. The associated proteins were detected by western blotting using anti-Flag antibody. (B) Whole-cell extracts from U2OS cells previously transfected with expression vector for p53 were incubated with GST fusion proteins of HEY1, the mutant HEY1-S68A, or HEY1-S68D coupled with Sepharose beads. The associated proteins were detected by western blotting using anti-p53 antibody.