Cell Lysate Preparation and Protein-Interaction Assays

Cell lysates were prepared from 10-cm dish cultures (1.5~2 × 10⁶ cells) with lysis buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 250 mM NaCl, 10% Glycerol, 5 mM EDTA, 1× complete protease inhibitor, 1× PhoSTOP) supplemented with detergents as follows. For protein-protein interaction assays, 0.2% Triton X-100 and 0.3% NP-40 were added. The resulting lysates were used for immunoprecipitation as previously described^{62 (link)}. For protein-RNA interaction, 0.5% Triton X-100 with 0.1% NP-40 was added. Recombinant NLRX1 was generated as previously described^{63 (link)}. Lysates were mixed with an equal volume of 2× incubation buffer (300 mM KCl, 40 mM HEPES, pH7.5, 10% glycerol, 200 Units/ml RNaseOUT (Thermo Fisher), 10 mM magnesium acetate, 2mM DTT). For precipitation, 500 ng biotin-labeled HAV RNA, 100 ng biotin-labeled poly(I:C) (InvivoGen) or 1 µl polyclonal anti-NLRX1 was used in each reaction. All reactions were gently rotated at 4 °C for 2 h, followed by the addition of magnetic streptavidin T1 beads (Invitrogen) for another 30 min rotation or protein G-Sepharose (GE Healthcare) for another 1 h rotation. After 5 intense washes with 1× incubation buffer, the final product was analyzed by qRT-PCR and/or immunoblotting.