ST-CH, ST-LGP2, and ST-LGP2–C615A cells (5 × 107) were plated and harvested after 24 hours. The cells were washed twice with cold PBS and lysed in 6 ml of lysis buffer [20 mM Mops-KOH (pH 7.4), 120 mM KCl, 0.5% Igepal, and 2 mM β-mercaptoethanol] supplemented with RNasin (200 U/ml; N2515, Promega) and cOmplete Protease Inhibitor Cocktail (#11873580001, Roche). Cell lysates were incubated on ice for 20 min with gentle mixing every 5 min and then clarified by centrifugation at 16,000g for 15 min at 4°C. Cell lysates were incubated for 2 hours on a spinning wheel at 4°C with 200 μl of StrepTactin Sepharose High Performance beads (#28-9355-99, GE Healthcare). For the RNAse treatment assay, RNaseA (#EN0531, Thermo Fisher Scientific) and RNaseIII (#M0245 L, NEB) were used at final concentrations of 0.1 mg/ml and 5 U/ml, respectively. Beads were collected by centrifugation (1600g for 5 min at 4°C) and washed twice for 5 min on a spinning wheel with 5 ml of washing buffer [20 mM Mops-KOH (pH 7.4), 120 mM KCl, and 2 mM β-mercaptoethanol] supplemented with cOmplete Protease Inhibitor Cocktail. Precipitates were eluted using biotin elution buffer (#2-1019-025, IBA). For the MS analysis, protein complexes were precipitated in 12% of trichloroacetic acid (TCA) (#T0699, Sigma) after 16 hours at 4°C. The dsRNA control for RNaseIII digestion encompassing 425 nt of MV genome sequence (L2) was obtained as previously described (66).

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