FIG. 2.

RIG-I sensor, but not Mda-5, triggers signaling events controlling p65Ser536 phosphorylation. A549 were transfected with control (Ctrl) (A to D), RIG-I (A and B), Mda-5 (A and C), or MAVS (D) siRNA oligonucleotides (Table 1) as described in Materials and Methods. (A) siRNA-transfected A549 cells were further transfected with the P2(2×)TK-pGL3 NF-κB firefly luciferase and the pRL-null Renilla luciferase (internal control) reporter constructs and either mock or RSV infected for 16 h. The luciferase activities were measured and are expressed as the fold activation over the nonstimulated cells after normalization with Renilla luciferase activities. (***, P < 0.001; mean ± the SEM; n = 6.) The data are representative of three different experiments. (B) WCE derived from siRNA-transfected A549 cells infected with RSV at an MOI of 3 for various periods were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted (IB) with anti-IκBαSer32 phospho-specific (IκBα-P-Ser32), anti-IκBα, anti-p65Ser536 phospho-specific (p65-P-Ser536), anti-p65, anti-RSV (nucleocapsid protein [N] is shown), anti-RIG-I (A), anti-MDA-5 (B), anti-MAVS (C), and anti-actin antibodies. The data are representative of three independent experiments.