Interaction in vitro; bait present on live, transfected S2 cells; prey produced from conditioned media of transfected mammalian 293T cell line.

The wntD-fz4 binding affinity was measured at 51 nM.

wntD bound to fz4 but not fz, fz2 or fz3 in this assay.

Putative receptor ligands were fused to nrt and transfected into S2 cells, the nrt protein acting to anchor putative ligands to the cell surface. Extracellular domains of putative receptor domains were fused to alkaline phosphatase, expressed in 293T cells and immunopurified from conditioned medium. Live cells expressing nrt-fusion proteins were mixed with alkaline phosphatase fusion proteins, and cell surface bound alkaline phosphatase was measured.