Figure 4.

In vitro and cell culture activities of RIG-I on short duplex RNAs. (A) The K_m,ATP of RIG-I stimulated by a library of duplex RNA constructs at ATP concentrations varying between 0 and 5 mM. (B) The K_m,RNA of RIG-I stimulated by a library of duplex RNA constructs at RNA concentrations varying between 0 and 500 nM. The K_m,RNA for the GC8 duplex is ∼100 nM and did not fit on the scale. (C) The k_cat summary averaged from the K_m,ATP and K_m,RNA experiments. (D) RIG-I stimulated IFN-β production was measured in 293T cells. RIG-I was stimulated by 5′triphosphorylated hairpins (20–650 nM) and the positive controls, poly I:C (15–500 ng/well) and 5′pppGC22 (20–650 nM). The increase in RNA concentration is indicated by a darkening color gradient. The relative luciferase is the firefly luciferase (IFN-β reporter) divided by the constitutively expressed Renilla luciferase. Error bars for the ATPase data report the standard deviation from at least three measurements. Error bars for the cell culture data report the standard error of the mean from three measurements. IFN-β, interferon-β; RIG-I, retinoic acid-inducible gene-I.