Supplementary Figure 1: Several WNTs bind to the extracellular domains of PKD1.

(a) HEK293T cells were co-transfected with indicated plasmids. Flag-tagged proteins were immunoprecipiated with rabbit α-Flag and immunoblotted with α-WNT5A (upper panel) or a combination of mouse α-Flag and mouse α-PKD1 (7e12) (middle panel). (b) HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-WNT5A (upper panel) or α-Fc (middle panel). (c) HEK293 cells stably expressing Fc, LRR-WSC-Fc, LDL-A-Fc, were co-cultured with HEK293 cells stably transfected with myc-tagged WNT5A for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-WNT5A (upper panel) or α-Fc (middle panel). Red or blue arrow heads indicate Fc fusions or WNT5A, respectively. (d) HEK293T cells were transiently transfected with F-BAP (lane 1) or full length F-PKD1 and TRPP2 (lane 2). Cells were separately transfected with WNT5A and co-cultured with F-BAP- or F-PKD1/TRPP2-transfected cells for 48 h. Flag-tagged proteins were immunoprecipitated with rabbit α-Flag and immunoblotted with α-WNT5A (lanes 1 and 2). Input WNT5A is shown in lanes 3 and 4. (e) Interaction of WNT4 with the LRR-WSC and LDL-A domains of PKD1. HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-WNT4 (upper panel) or α-Fc (middle panel). (f) Interaction of Flag-tagged WNT3A with the LRR-WSC domain of PKD1. HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-Flag (upper panel) or α-Fc (middle panel). (g) Myc-tagged human TRPP2 was purified from transiently transfected HEK293T cells using α-Myc and incubated (∼30 ng) with 1 μg ml⁻¹ of purified WNT9B. Immobilized myc–TRPP2 was immunoblotted with α-WNT9B. Experiments shown in (a) and (e) were done once. Experiments shown in (d) and (g) were successfully repeated twice. Experiments shown in (b), (c), and (f) were repeated 3 times. Uncropped western blots are provided in Supplementary Fig. 8.