eIF-4E was purified by m[7]GTP-sepharose chromatography, and eIF4G copurified.

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

Proteins bound to m[7]GTP-sepharose column was fractionated by density gradient sedimentation.

From partially purified extracts, eIF-4E and eIF4G cosediment through density gradient.

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

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

Positive control.

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

Interaction in vitro; bait produced and labeled by in vitro translation; prey produced and labeled by in vitro translation.

cup and @ compete for binding to eIF-4E@.

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 cell extract of S2 cell line; bait produced from endogenous gene; prey produced from endogenous gene.

The eIF4E1-eIF4G1 interaction decreases by treatment with increasing amounts of Thor.

Bait endogenous to testis extract.

Prey endogenous to testis extract.

Positive control.

eIF4G binds eIF-4E but not eIF-4E-6 in this assay.

CG2950 (mxt) exhibited the strongest affinity for eIF-4E, compared to eIF4G and Thor.

This interaction persists in the presence of RNaseA.

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

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

Kd = 17 nM

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

eIF4E1 was pulled down using m[7]GTP-sepharose beads.

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

Addition of Thor aa 50-83 disrupted eIF4E1-eIF4G1 interaction. R56E and R63E mutations in Thor were unable to disrupt eIF4E1-eIF4G1 interaction and S65D, T70D linker domain mutations resulted in less efficient disruption. .