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Differential protease sensitivity of active and inactive MDA5 variants. His6-tagged MDA5 wild type, motif I, and motif II mutant proteins were expressed in Sf9 insect cells by infection with recombinant baculovirus and affinity-purified. Proteins were subjected to limited digestion with chymotrypsin in a 1:1000 enzyme:substrate ratio for the indicated times. Samples were analyzed by immunoblot using MDA5-specific antiserum. FL, full-length MDA5; F1, fragment 1; F2, fragment 2.
