For single-molecule analysis, lysate from S2 cells, in which pAHisHaloW-Dcr-2 wild-type, G31R, or D1217A/D1476A was overexpressed, was incubated with 0.5 µM HaloTag Cy5-biotin ligand31 (link) for Dcr-2-tethered experiments, or 35 µM HaloTag Alexa fluor 660 ligand (Promega) for dsRNA-tethered experiments, at 25 °C for 30 min. The labeled proteins were subjected to SDS-PAGE and visualized by a LAS-3000 image system (Fujifilm). Free ligands were removed by purification using cOmplete His-Tag Purification Resin (Sigma-Aldrich), and the labeled proteins were supplemented with 10% glycerol, 0.2 mg/ml BSA, shock-frozen, and stored at −80 °C.
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