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

Cells expressing fz were incubated with wg-conditioned medium, then fixed and analyzed for wg surface binding by immunofluorescence.

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

The wg-fz binding affinity was measured at 45 nM.

wg bound to fz, fz2 and fz3, but not fz4 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.

fz expressed in cells and mixed with wg-containing conditioned medium; binding to cell surface detected by immunofluorescence using wg antibody.

Positive control.

Bait expressed in transiently transfected cells.

Prey expressed in stably transfected cells.