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

Purified proteins were mixed and 1:1 complexes were isolated by size-exclusion chromatography, with an elution profile corresponding to the 58kDa complex.

Two-hybrid system: yeast

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

Dhit was indentified in independent screens using wild-type and activated (Q205L) versions of Gαo as bait.

Gαo was matrix-immobilized on CNBr-activated sepharose, pre-loaded with GDP, GTPγS or AIF[[4]][-], then incubated with Dhit.

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

Full length and N-terminal portions of Dhit bind to Gαo regardless of its nucleotide state; the C-terminal, RGS domain-containing portion of Dhit preferentially binds to activated (GTPγS-bound) Gαo.

Both N-terminal and C-terminal portions of Dhit can bind to Gαo, and increasing amounts of N-terminal Dhit do not prevent binding of C-terminal Dhit to Gαo.

Dhit is outcompeted by purified porcine Gβγ for binding to Gαo.

Dhit was matrix-immobilized on CNBr-activated sepharose, then incubated with head extract.

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

Dhit can pull down activated, GTPase-deficient Gαo from transgenic head extracts, but not the wild-type Gαo (which is presumably sequestered by Gβγ).