Interaction in vitro; bait produced as a recombinant fusion protein in COS1 cell line; prey derived from conditioned medium of cell line.

hh binds the first FNIII domain of ihog, but not its second FNIII domain.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

The interaction of purified ihog and hh occurs only in the presence of heparin.

In the presence of heparin, hh and ihog form complexes having stoichiometries of up to 2:2 .

Native molecular weight of complexes were determined by size exclusion chromatography of purified proteins.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

Molecular weight of complexes estimated by analytical ultracentrifugation and calculation of sedimentation coefficients.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

A mixture of hh and ihog crystallized in the presence of heparin, but well ordered heparin was not apparent in the crystal structure.

hh and ihog form a heterotetramer complex with a 2:2 stoichiometry.

The structures of hh and ihog within the complex are fundamentally unchanged from their unbound conformations.

Interaction in vitro; bait produced from transfected construct; prey produced from transfected construct and collected in conditioned medium.

Heparinase treatment of cells reduces the interaction of hh with ihog in this assay.

Heparin is hypothesized to bind positively charged surfaces on hh and ihog to bridge the interaction.

The binding of hh to ihog-expressing cells is increased by co-expression of ptc.

Luciferase-tagged hh was mixed with intact, washed cells expressing ihog.

Source was cell extract of S2R+ cell line; bait produced from transfected construct; prey produced from transfected construct.

ihog and hh were expressed in separate cells, then hh was added to the external medium of ihog transfected cells before coimmunoprecipitation.

Interaction in vitro; bait produced as a recombinant fusion protein in baculovirus system; prey produced as a recombinant fusion protein in unspecified cell line.

High affinity binding of hh requires both ptc and ihog.

Cell-free binding of hh to vesicles containing ptc and/or ihog.

Interaction in vitro; bait produced as a recombinant fusion protein in unspecified cell line; prey produced as a recombinant fusion protein in S2R+ cell line.

hh was bound to beads then mixed with detergent-solubilized extracts of transfected S2R+ cell line.

Positive control.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey produced as a recombinant fusion protein in bacterial system.

Proteins were purified by ion-exchange and size-exclusion chromatography for use in this assay.

The hh-ihog was not detected in the absence of heparin.

The binding affinity of ihog for hh was Kd = 1.77E-6 M in the presence of 1uM heparin.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey produced and labeled by in vitro translation.

Source was larval wing discs of transgenic fly line; bait produced from transgenic construct; prey produced from transgenic construct.

Bait protein was over-expressed in the dorsal compartment with the ventral compartments as an internal control. An additional experiment fused the FNIII-1 domain to the extracellular domain of CD8 to target it to the membrane. The ability of the bait to recruit prey protein to the ventral compartment was taken as evidence of direct physical interaction.