3.3 ISX9 potentiates the association of LRP6 with Axin1
It has been known that Wnt stimulation-induced LRP6 phosphorylation promotes the association of LRP6 with Axin, a key step to regulate β–catenin stability (Xin Zeng et al., 2008). We therefore examined the effect of ISX9 on interaction between LRP6 and Axin1 using a mammalian two-hybrid assay consisted of UAS-TK-Luc reporter and the expression vectors for Gal4-LRP6 intracellular domain (Gal4-LRP6-ICD) and VP16-Axin1. The interaction between LRP6-ICD and Axin1 was observed in HEK293T cells transfected with both Gal4-LRP6-ICD and VP16-Axin1, and treatment with ISX9 dose-dependently potentiated the interaction between LRP6-ICD and Axin1. However, little signal was detected when UAS-TK-Luc reporter was co-expressed with either Gal4-LRP6-ICD or VP16-Axin in the presence or absence of ISX9 (Figure 3A). Notably, LiCl treatment exerted little effect on the association of Gal4-LRP6 ICD with VP16-Axin1 (Figure 3B). These results suggest that ISX9 may potentiate the association of LRP6 with Axin1. To further confirm the effect of ISX9 on the interaction of LRP6 with Axin1, a co-immunoprecipitation assay was performed using anti-Flag beads in HEK293T cells transfected with LRP6-V5 and Flag-Axin1 expression vectors. Figure 3C showed that increasing amounts of LRP6-V5 protein were immunoprecipitated using anti-Flag beads in the presence of ISX9 (Figure 3C). Treatment with ISX9 also promoted endogenous interaction between LRP6 and Axin1 in HEK293T and HaCat cells (Figure 3D and E). These results demonstrated that ISX9 is capable of promoting the interaction of LRP6 with Axin1.