FIG 4 .

Rig-I ATPase activity promotes RNA recycling. (A) RNA pulldown assays were performed using 13 pmol of biotinylated (Biot.) 10r-10d and 20r hybrids (as indicated) and purified his-RIG-I (60 nM) in the presence of ATP and absence of MgCl₂. Competition experiments were performed by adding 52 pmol of the same non-biotinylated RNA molecule (competitor) to the reaction mix in the presence or absence of MgCl₂ (as indicated). (B) RNA pulldown assays were performed using 13 pmol of biotinylated 20r hybrids and purified his-RIG-I (60 nM) in the absence of ATP and MgCl₂. Competition experiments were performed by adding 52 pmol of the same non-biotinylated RNA molecule (competitor) to the reaction mix in the presence or absence of MgCl₂ (as indicated). The amounts of RIG-I remaining on the beads were determined by Western blotting using an anti-RIG-I antibody and quantified. RIG-I quantifications are indicated as the average percentage ± SD. (A) n = 3 for 10r-10d/RIG-I wt; n = 6 for 20r/RIG-I wt and 20r/K270A. (B) n = 5 for 20r/RIG-I wt; n = 3 for 20r/K270A.