Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from embryo cDNA expression library).

Two-hybrid system: yeast GAL4-BD/VP16-AD

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from transfected 293T cell extract.

The WW2 and WW3 domains of Nedd4 (but not WW1) can interact with comm. Both comm PY motifs (PPCY and LPSY) must be mutated to abolish binding in this assay (only the LPSY motif is necessary in two hybrid assays).

Source was yeast cell line; bait produced as transgenic fusion protein; prey was previously cloned reagent.

Two-hybrid system: yeast GAL4-BD/GAL4-AD

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

A catalytically inactive form of Nedd4 was tested in this assay.

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

Mutation of both comm PY motifs (PPCY and LPSY) disrupts binding to Nedd4 (but not to robo1).

A catalytically inactive form of Nedd4 was tested in this assay.

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

This assay detected no decrease in binding affinity for a H237A mutation in the LPSY sequence of comm. Additionally, a longer version of the LPSY sequence from aa 225-239 showed similar binding affinity as the shorter sequence. A peptide consisting of aa 216-227 did not bind Nedd4.

Source was embryos of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.

comm-robo1-Nedd4 form a complex. Captured by 3 pairwise interactions.