Fig. S6

Waif1 does not interfere with LRP6-Frizzled8 interaction, but blocks Dkk1-dependent LRP6 internalization, related to figure 6.

(A-B) The Waif1a extracellular domain (ED) does not directly bind the LRP6 ED and does not interfere with Wnt1-dependent interaction of the LRP6 ECD and Fzd8 CRD domain.

(A) In mixtures of conditioned media, LRP6N-myc co-immunoprecipitates with Dkk1-IgG (lane 5), but not beyond background levels with Waif1aN-IgG (lane 6).

(B) In mixtures of conditioned media, LRP6N-myc co-immunoprecipitates with the CRD domain of Fz8 in the presence (lane 3), but not the absence of Wnt1 (lane 2). Dkk1-Flag interferes with the LRP6- Fz8CRD interaction (lane 4), but Waif1N-GFP does not (lanes 5-6).

(C) Waif1a does not block Wnt induced LRP6 phosphorylation. Overexpression of Waif1a-Flag in MEFs does not reduce Wnt3a-induced LRP6 phosphorylation, which is already apparent 15 minutes after stimulation with Wnt3a CM.

(D) Human Waif1 inhibits pBAR activity in HEK293T cells induced by cotransfected Wnt8, but not by constitutive active LRP6ΔN, while dominant-negative TCF3 (ΔNTCF3-myc) can suppress LRP6ΔN induced pBAR activation. ***, p<0.001 Student′s t-test.

(E-G) Waif1a specifically interferes with Dkk1-induced LRP6 internalization.

(E) Waif1a interferes with Dkk1-mediated internalization of LRP6 (arrow). HEK293T cells stably expressing LRP6-GFP were transfected with mRFP-GPI or Waif1a-Cherry, incubated with Dkk1 conditioned media at 4°C, left there (upper panel) or shifted to 37°C and imaged 30 minutes later. Scale bar 10μm.

(F) Quantification of Dkk1-induced LRP6 internalization data presented in K. n (cells with LRP6 puncta) = 360, n (mRFP-GPI) = 81, n (Waif1a-Cherry) = 12. ***, p<0.001 Chi-Square test.

(G) Waif1a does not inhibit Clathrin-dependent internalization of Transferrin. HEK293T cells transfected with the indicated constructs were incubated with fluorescently labeled Transferrin for 10 min, washed and imaged. Note that treatment with the dynamin-inhibitor Dynasore blocked Transferrin uptake, while Waif1a-GFP did not. Scale bar 10μm.

(H-I) Waif1a enhances localization of LRP6 to the plasma membrane in Xenopus animal cap cells, which express dkk1.

(H) Apotome images of Xenopus animal cap explants injected with 60 pg lrp6-HA, together with 200 pg waif1a-GFP or in addition with 100 pg dkk1-Flag. Note increased membrane staining of LRP6-HA in the presence of waif1a, which is partly reversed by dkk1.

(I) Quantification of LRP6 localization shown in L.

(J) Waif1a and LRP6 compete for Dkk1 binding. Immunoprecipitation of LRP6-HA from HEK293T cells co-IPs Dkk1-Flag (lane 4), but much less so in the presence of Waif1a-GFP (lane 5). Conversely, significantly more Waif1a-GFP binds to LRP6-HA in the absence of Dkk1-Flag (compare lanes 5 and 6).