2.7 Microscale thermophoresis
Microscale thermophoresis (MST) experiments were conducted on a Monolith NT.115 system (NanoTemper Technologies GmbH, Germany), which was used to quantify the interaction between Axin1 fragments (Axin1 1-300, Axin1 301-521, Axin1 522-862) and ISX9, and the interaction between LRP6-ICD and ISX9, respectively. Axin1 fragments or LRP6-ICD were labeled with the manufacturer’s labeling kits (MonolithTM RED-NHS, NanoTemper Technologies GmbH, Germany). During the measurement of Axin1 fragments with ISX9, the concentration of labeled Axin1 fragments or LRP6-ICD is 100 nM. Unlabeled ISX9 was prepared in 10 mM PBS dimethyl sulfoxide (DMSO), with the final concentrations of ISX9 ranging from 0.61 nM to 5 μM, the samples were added to the monolith capillaries (MO L022, NanoTemper Technologies) and subsequently subjected to MST analysis. The values obtained were normalized and plotted against the Axin1 fragments or LRP6-ICD and ISX9 concentration, respectively. The dissociation constant was then determined using a single-site model to fit the curve.