Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type adult head extract.

Extracts were fractionated by ion exchange then gel filtration chromatography. The presence of shi-binding protein was monitored by far western blotting. THe purified protein was identified by peptide sequencing.

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

Adult head extract was separated by gel filtration, and fractions containing Dap160 in complexes of about 500 kDa were used for immunoprecipitation.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type adult head extract.

Prey identification based on comigration of far western band with band detected by immunoblot using anti-shi antisera.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type or transgenic adult head extract.

The SH3B domain of Dap160 is the key shi binding module, but constructs with SH3 domains A-B or SH3 domains A-D display stronger binding.

The shi-Dap160 interaction is increased in a mnb mutant background.

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

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type adult head extract .

The SH3B domain, located within amino acids 732-834 of Dap160, is the major binding module for the interaction between Dap160 and shi. The domains SH3A and SH3B of Dap160 also bind shi in an additive fashion. The binding of both Dap160 SH3A and B domains, and the B domain alone, is not different than the binding of all four SH3ABCD domains. Additionally, the binding of all four Dap160 SH3ABCD domains decreases under phosphorylation-promoting conditions.