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

The pins GoLoco domain (aa 372-658) does not pull down mud in this experiment.

Interaction in vivo; bait derived from wild-type and transgenic embryonic extracts; prey derived from wild-type and transgenic embryonic extracts.

Extracts from flies over expressing pins constructs were used to precipitates mud. Extract from wild type animals did not precipitate mud.

pins protein has three Gαi binding sites. In this paper, each of these binding sites is disrupted via point mutation and the subsequent mud binding activity of pins is assayed, demonstrating that these sites, and by extension, Gαi binding is important to the pins - mud interaction.

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

Similar experiments were carried out in a ctp mutant background and an ana2 mutant background. The interaction between mud and pins was severely reduced in these backgrounds.

Source was larvae of wild type fly line; bait produced from tagged endogenous gene; prey produced from endogenous gene.

The interaction between mud and pins was not affected by increasing concentrations of Pen or Fs(2)Ket.

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

Given that raps coimmunoprecipitates with insc and mud, but insc and mud do not coimmunoprecitate each other, the authors conclude that raps forms mutually exclusive complexes with insc and mud.

Bait endogenous to larval brain extract.

Prey endogenous to larval brain extract.

Given that raps coimmunoprecipitates with insc and mud, but insc and mud do not coimmunoprecitate each other, the authors conclude that raps forms mutually exclusive complexes with insc and mud.

Bait endogenous to larval brain extract.

Prey endogenous to larval brain extract.

GST pull down.

Bait expressed and purified from bacteria.

Prey expressed and purified from bacteria.

mud and insc compete for binding to raps.

GST pull down.

Bait expressed and purified from bacteria.

Prey expressed and purified from bacteria.

Excess insc disrupts the interaction of raps with mud, consistent competition between mud and insc for raps binding.

Bait peptide synthesized, fluorescently labeled and reverse-phase HPLC purified.

Prey expressed and purified from bacteria.

The interaction of raps with mud is inhibited by insc protein, which does not itself interact with mud.