Western blot analysis of WT NS5 binding to biotinylated 5′SLA RNA following coimmunoprecipitation. (A) WT NS5 was incubated with immobilized biotinylated 5′SLA. After excess unbound NS5 was removed, increasing amounts of 3′SL were added (left). Quantitation of the NS5 band was performed using ImageJ and normalized to 100% for the sample with no added 3′SL (right). (B) WT NS5 was incubated with immobilized biotinylated 5′SLA as in A, but increasing amounts of 3′SL were added without removal of excess unbound NS5 (left). Quantitation of the NS5 was done as in (A) (right). Molar ratio of 3′SL to 5′SLA is shown on top of the gel image, and NS5 was detected using the 5M1 antibody. The statistical significance of the difference between two groups was evaluated by Student's t-test and a P-value <0.05 was considered significant (≤0.0001, ≤0.001, ≤0.01, ≤0.05 were represented by [****], [***], [**], [*], respectively).
