FIGURE 2.
Mutations to RNA helicase motifs eliminate ATP hydrolysis activity. FLAG-tagged wild type and mutant MDA5 (A), RIG-I (B), and LGP2 (C) proteins were expressed in HEK293T cells and immunoaffinity-purified with FLAG M2 affinity gel. Eluted proteins were incubated with [γ-32P]ATP in the presence or absence of poly(I-C) and separated by thin layer chromatography (top). Origin and migration of free phosphate (Pi) and ATP are indicated. ATP hydrolysis activity was quantified by phosphorimage analysis and plotted as percentages of wild type protein activity in presence of poly(I-C) (center). Bottom panels show silver-stained SDS-PAGE demonstrating similar amounts of purified proteins in reactions. GFP, green fluorescent protein.