Fig. 4.

Enhancers potentiate binding and ubiquitylation of the unphosphorylated β-catenin degron. a Binding of Ser33/S37A β-catenin peptide for β-TrCP measured in the TR-FRET assay in absence or presence of 20 μM NRX-252114. For reference, the binding curve for pSer33/pSer37 β-catenin peptide in the same TR-FRET assay is represented in dotted gray line. b Ubiquitylation of Ser33/S37A β-catenin peptide (residues 17–60) by SCF^β-TrCP in the presence of NRX-252114 under conditions similar to Fig. 3d. c 400 nM purified full-length S37A mutant β-catenin protein was ubiquitylated in the presence of 125 nM Ube1, ATP, 1.75 μM Cdc34, and 400 nM SCF^β-TrCP with the increasing concentration of NRX-252114. Reactions were resolved by SDS-PAGE and analyzed by immuno-blotting with a β-catenin C-terminal antibody. For reference, ubiquitylation of WT β-catenin protein is also shown (* refers to degradation products)