Fig. 11.
Effect of AGR2 peptide docking mutants binding to EpCAM. A, His-EpCAM was immobilized onto the well surface of a microtiter plate. Titration of AGR2 WT and mutants (0–0.5 μg) were added in mobile phase and AGR2 binding without (A) and with 1 mm DTT (B) to immobilized EpCAM was quantified using a specific AGR2 antibody. The binding is plotted as the extent of protein-protein complex formation in RLU as a function of increasing protein in the mobile phase. Reactions in (A) have no DTT included in the AGR2-binding reactions and (B) includes DTT at the stage of addition of AGR2 binding to reveal any effects of potential cysteine oxidation on protein-protein interactions (as highlighted in C where both AGR2 and EpCAM should be in the reduced state). D, Staging the effects of DTT by including reductant in the blocking step to determine whether the DTT effect (from B) on the reaction is because of EpCAM substrate and not AGR2 itself. E, A titration of wt-AGR2 and AGR2S134A in the presence or absence of reductant in the blocking stage. As in (A and B), the binding is plotted as the extent of protein-protein complex formation in RLU as a function of increasing protein in the mobile phase.