Figure 3.

RIG-I is stimulated by the ends of poly I:C. (A) LMW poly I:C was fractionated on an analytical Superdex 200 size exclusion chromatography column and separated into seven fractions on a 15% polyacrylamide, 4M urea semi-denaturing gel stained with ethidium bromide (marker is in base pairs). (B) ATPase activity of RIG-I stimulated by 0–15 ng/μl of the poly I:C fractions A1, A3, A5 and A7 at 5 mM ATP. The data were fit to the quadratic form of the Briggs–Haldane equation with the assumption that the k_cat values are the same for all the fractions. (C) The K_m,ATP for RIG-I stimulated by 15 ng/μl of the poly I:C fractions A1–A7 while varying the ATP concentration from 0–5 mM ATP. (D) The calculated K_m,RNA for RIG-I stimulated by 0–15 ng/μl of the poly I:C fractions A1–A7 at 5 mM ATP. The K_m values in the left panel are in ng/μl and values in the right panel are in nM for all seven fractions on the basis of the estimated sizes of each fraction. Error bars for the poly I:C data report the standard deviation across six experiments. LMW, low molecular weight; RIG-I, retinoic acid-inducible gene-I.