Proteins were assayed for cooperative DNA binding to [32]P labeled EcR DNA element from Hsp27 promoter in gel shift assays. Neither usp or EcR bind to DNA alone.

The presence of each usp and EcR in the protein:DNA complex was confirmed by using antibodies to bind and supershift the protein:DNA complex.

Interaction in vitro; proteins produced by in vitro translation.

usp and/or EcR were mixed with [32]P labeled Hsp27 ecdysone-response element DNA. DNA-protein complexes observed by EMSA were used to deduce protein-protein interactions.

usp and EcR bind the DNA probe cooperatively; alone, neither was observed to bind DNA.

Interaction in vitro; proteins produced by in vitro translation.

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

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

S2 nuclear extract was mixed with [32]P labeled Hsp27 DNA, then treated with anti-usp or anti-EcR antibodies to detect each protein in the DNA-protein complex (detected as decreased mobility (super shift) of the observed EMSA band).

Together, usp and EcR form a DNA-protein complex with Hsp27 DNA.

Interaction in vitro; proteins derived from S2 nuclear extract.

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

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

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

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

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

Two-hybrid system: yeast GAL4-BD/GAL4-AD. Treatment with ecdysone receptor agonist, Muristerone A, increases the usp-EcR interaction.

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

Two-hybrid system: yeast GAL4-BD/GAL4-AD. Treatment with ecdysone receptor agonist, Muristerone A, increases the usp-EcR interaction.

Interaction in vitro; components produced as recombinant fusion proteins in reticulocyte lysate system.

A [32]P ATP labelled 80-bp DNA fragment containing a single GAL4 binding site was used as a DNA target for this experiment.

proteins were crystallized with IR-1 DNA.

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

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

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

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

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

Muriateron A enhances dimerization of EcR and usp.

Source was S2 cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

S2 cell Two-hybrid system: GAL4-BD/VP16-AD

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

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 was previously cloned reagent).

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 was previously cloned reagent).

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

Positive control.

Source was Drosophila melanogaster L57-3-11 cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

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

Luciferase reporter was used to assess two hybrid activity.

Positive control.

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

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

Positive control.

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

Interaction shown with EcR isoforms A and B1

Source was cell extract of S2R+ cell line; proteins produced from transfected construct or endogenous gene.

HGScore = 43.885422

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

Positive control.

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

Positive control.

Source was cell extract of S2R+ cell line; bait produced from transfected construct; prey produced from transfected construct.

Experiment carried out in cells expression EcR, usp and twi.