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.