Figure 5: Cholesterol binding of Dvl is required for canonical Wnt signalling under physiological conditions.

(a) Induction of the secondary axis by exogenous addition of XDvl2. The partial axis was characterized by the axial structure without the head structure, including eyes and cement glands. The complete axis has the axial structure along with the head structure. Scale bar, 1 mm. (b) Quantification of Fig. 5a. (c) The TOP-flash activity was measured in Xenopus AC explants. XWnt8 mRNAs were injected with indicated XDvl2 mRNAs and XDvl Morpholinos (MO). At stage 10, ACs were dissected and analysed. Error bars indicate s.d. from three independent analyses. (d) The western blot analysis of activated (dephosphorylated) β-catenin levels. α-Actin was used as a loading control. (e) Western blot data showing the degree of LRP6 phosphorylation. Xenopus embryos were injected with indicated mRNAs, including human Myc-tagged LRP6 (hLRP6-Myc), and AC tissue explants were analysed at stage 10.5. Only exogenous Dvl2 WT could rescue Dvl MO-mediated inhibition of LRP6 phosphorylation in Xenopus AC tissues. hLRP6-Myc was immunoprecipitated by the c-Myc antibody and phosphorylated LRP6 (pLRP6) was detected with the antibody against Sp1490. (f) Confocal images of HeLa cells showing the location and degree of LRP6 phosphorylation. The Wnt3a-mediated co-localization (see white arrows) of GFP-human LRP6 (hLRP6) (green) and phosphorylated LRP6 at Ser1490 (pLRP6: red) was clearly seen at the PM and endosomes in control cells (Con short interfering RNA (siRNA): first row). Dvl siRNA (second row) abrogated the co-localization of hLRP6 and pLRP6, which was significantly rescued by XDvl2 (third row), but not by XDvl2 FM/A (bottom row). Scale bar, 20 μm. (g) Quantification of Fig. 5f.