ATPase activity of MDA-5. (A) Electrophoretogram of purified proteins. GST and GST-MDA-5 were resolved on 9% SDS/PAGE and stained with Coomassie brilliant blue. (B) Effect of divalent metal ions and RNA concentration on MDA-5 ATPase activity. ATPase activity assay was performed with variable poly(I)⋅poly(C) concentrations (0.038, 0.375, 3.75, 37.5, and 375 ng/μl). MnCl2 (3 mM, ■) substituted for MgCl2 (3 mM, ♦). The results were quantitated by PhosphorImager. The data shown are the mean ± SD from two experiments. Specific activity is nmol/min/μg of protein. (C) Lineweaver–Burk plot of the effect of [ATP] on MDA-5 ATPase activity. ATPase activity was measured with various ATP concentrations (15.6, 31.3, 62.5, 125, 250, and 500 μM) for 20 min in a 37°C air incubator and quantitated by PhosphorImager. The result shown is the mean ± SD from three independent experiments. (D) Autoradiogram of MDA-5 ATPase assay with various RNA species. The indicated types of RNA (20 ng/μl) were added in standard reaction mixture, and the results presented were obtained by exposing a TLC plate to Kodak X-Omat film. D.W., distilled water.