Proteins immunopurified from embryonic high-speed supernatant.

pnut, Sep1 and Sep2 copurify in about a 1:1:1 stoichiometry.

Source was embryos of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Proteins immunopurified from embryonic high-speed supernatant using anti-pnut antibody, then subjected to gradient fractionation.

pnut, Sep1 and Sep2 cosediment as a complex at about 8S in a 1:1:1 stoichiometry.

Source was embryos of wild-type fly line; proteins produced from endogenous gene.

Proteins immunopurified from embryonic high-speed supernatant using anti-pnut antibody, then subjected to gel filtration chromatography.

pnut, Sep1 and Sep2 copurify as a complex at about a 1:1:1 stoichiometry.

Source was embryos of wild-type fly line; proteins produced from endogenous gene.

Two-hybrid system: yeast LexA-BD/B42-AD

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

6 Sep2 clones obtained in the pnut screen.

Two-hybrid system: yeast LexA-BD/B42-AD

8 pnut clones obtained from the Sep2 screen.

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

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

Sep1, Sep2 and pnut were coexpressed and purified by sequential His then Strep affinity purifications.

Source was cell extract of baculovirus-infected cell line; bait produced from transfected construct; prey produced from transfected construct.

Mutations to the GTPase domains of pnut (K155A, D206A, K288A) or Sep2 (K56A, D102A, K186A) do not affect the interaction.