RIG-I Enzymatic ATPase Activity Assay

The RIG-I ATPase assay was performed as previously described⁵⁵ . Briefly, 250 nM of in vitro–transcribed RNA were incubated with 100 nM of RIG-I protein purified from insect cells (as previously described^{19 (link)}) and 3 mM unlabeled ATP, including trace amounts of [γ-^{32 (link)}P]ATP in EMSA buffer (5 mM MgCl, 50 mM KCl, 50 mM HEPES, 1 mM TCEP, 0.1 mg/ml BSA, pH 7) for 0, 0.5, 1, 2, or 3 h at 37 °C. Free phosphate was separated from unhydrolyzed ATP by thin layer chromatography in TLC running buffer (1 M formic acid, 0.5 M LiCl) on polyethyleneimine cellulose TLC plates (Sigma-Aldrich). [γ-^{32 (link)}P]Pi and [γ-^{32 (link)}P]ATP were detected using a phosphor-imaging system (GE Healthcare), quantified using ImageJ, and the percentage of hydrolyzed ATP for every time point was calculated. The average value of three replicates was calculated and average hydrolyzed ATP was plotted against time.