S2 cell extracts were fractionated by ammonium sulfate precipitation, phenyl-sepharose, Mono S, Mono Q, hydroxyapatite, Mono S and gel filtration chromatography. siRNA-generating activity was tracked during fractionation until a fraction containing two bands of 190 kDa (Dcr-2) and 36 kDa (r2d2) was recovered. Bands were identified by mass spectroscopy.

The siRNA generating activity cofractionates with r2d2 and Dcr-2, but not with Dcr-1.

Source was cell extract of S2 cell line; proteins produced from endogenous genes.

S2 cell extract was immunodepleted for r2d2, and co-depletion of Dcr-2 was monitored by western blot.

Immunodepletion of r2d2 co-depletes Dcr-2 but not Dcr-1. Additionally, Dcr-2-directed RNAi destabilizes r2d2 protein; Dcr-1-directed RNAi does not affect r2d2 stability.

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

Purified r2d2 and Dcr-2 were incubated with [32]P labeled siRNA and RNA-binding was monitored by gel electrophoresis shift assay. The presence of r2d2 and Dcr-2 in the RNA:protein complex was monitored by supershift using anti-Dcr-2 and anti-r2d2 antibodies. Dcr-2 alone did not bind siRNA in this assay.

Interaction in vitro; bait produced as a recombinant fusion protein in baculovirus-infected Sf21 system; prey produced as a recombinant fusion protein in baculovirus-infected Sf21 system.

Embryonic lysate was cross-linked to [32]P labeled siRNA, immunoprecipitated, and proteins visualized by autoradiography.

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

Embryonic lysate was cross-linked to [32]P labeled siRNA, immunoprecipitated, and proteins visualized by autoradiography.

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

[32]P labeled siRNA was incubated with extract and UV crosslinked to RNA binding protein. The extract was then immunoprecipitated with r2d2 antibody and coprecipitated RNA binding proteins were visualized by SDS-PAGE and autoradiography. An observed 200 kDa band was judged to represent Dcr-2 based on the molecular weight and the band\'s absence in Dcr-2 mutant extracts.

r2d2 coimmunoprecipitates Dcr-2 but not Dcr-1.

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

Dcr-2 coimmunoprecipitates with r2d2 but not loqs (r3d1).

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

Extract was fractionated by sequential ammonium sulfate precipitation and phenyl-sepharose, Mono S, Mono Q and hydroxyapatite chromatography.

Dcr-2 and r2d2 copurify together.

Source was cell extract of S2 cell line; proteins produced from endogenous genes.

[32]P labeled siRNA was incubated with purified Dcr-2 and r2d2. The formation of RNA:protein complexes was monitored by gel shift assay.

Dcr-2 and r2d2 coordinately bind siRNA. Alone, neither has siRNA binding activity in vitro.

Interaction in vitro; proteins produced as a recombinant fusion protein in baculovirus-infected insect cell system.

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

Positive control.

r2d2 interacts with Dcr-2 but not Dcr-1.

In embryos, anti-Dcr-2 coimmunoprecipitates r2d2 but not loqs.

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

In adults, anti-Dcr-2 coimmunoprecipitates r2d2 but not loqs.

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

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

Interaction observed in both nuclear and cytoplasmic extracts.

Dcr-2 and r2d2 were co-expressed, purified by gel filtration and mixed with the let-7 siRNA duplex. The ternary complex was column purified and detected by cryo-EM.

Interaction in vitro; proteins produced as a recombinant fusion proteins in baculovirus system.

Interaction in vitro; proteins produced as a recombinant fusion proteins in a baculovirus and insect cell system.

Higher order complexes consisting of multiple Dcr-2 and r2d2 proteins, in addition to loqs bound to siRNA, curated as a series of binary protein-protein interactions.

Higher order complexes consisting of multiple Dcr-2 and r2d2 proteins, in addition to loqs bound to siRNA, curated as a series of binary protein-protein interactions.

Interaction in vitro; proteins produced as a recombinant fusion proteins in a baculovirus and insect cell system.

Interaction in vitro; proteins produced as a recombinant fusion proteins in a baculovirus and insect cell system.

Ternary complex consisting of Dcr-2, loqs and r2d2 curated as a series of binary protein-protein interactions.

Higher order complexes consisting of multiple Dcr-2 and r2d2 proteins, in addition to loqs bound to dsRNA, curated as a series of binary protein-protein interactions.

Interaction in vitro; proteins produced as recombinant fusion proteins in a baculovirus and insect cell system.