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

Roughly 80% of fz2-transfected cells show surface binding of wg.

Cells expressing fz2 were incubated with wg-conditioned medium, then fixed and analyzed for wg surface binding by immunofluorescence.

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

Cells expressing fz2 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-fz2 binding affinity was measured at 54 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.

Interaction in vitro; bait produced from transfected construct and present on surface of S2R+ cells; prey produced from conditioned medium of stably transfected cell line.

Cells expressing fz2 were incubated with wg-conditioned medium, then fixed and analyzed for wg surface binding by immunofluorescence.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from transfected construct.

Cotransfecting increasing amounts of dlp increases the fz2-wg interaction presumably by stabilizing wg protein up to a certain point. Adding large amounts of dlp decreases the fz2-wg interaction presumably by competing with fz2 for dlp binding.

Purified fz2 protein was bound to plate, exposed to purified wg protein, and binding was detected using wg primary and alkaline phosphatase-conjugated secondary antibodies.

Positive control.

Bait expressed and purified from cells.

Prey expressed and purified from stably transfected cells.

Binding affinity of wg-fz2 interaction: 1.3 nM.