Fig. 3.

ATP hydrolysis promotes dissociation of MDA5, but not RIG-I, from dsRNA at a rate inversely proportional to dsRNA length. (A) Schematic of pull-down dissociation kinetic assay. The level of MDA5 bound to biotinylated dsRNA was monitored at discrete time points during dissociation by using streptavidin magnetic bead pull-down. MDA5 was fluorescently labeled for quantitation on SDS/PAGE. See Fig. S3 for details. (B) A representative SDS/PAGE image of MDA5 (Left) or RIG-I (Right) from dissociation pull-down assays at t = 0 or 2 min (2 m) using 512-bp dsRNA with either ATP or ADPCP (2 mM). Nonspecific binding (BG) was measured by using nonbiotinylated dsRNA. (C) Analysis of dissociation kinetics of MDA5 with ATP or ADPCP. Biotin pull-down was performed as in B using dsRNAs of 112–2,012 bp (mean ± SD, n = 3).