FIG. 3.

Downregulation of TRAF6, but not of TRAF2 or TRAF3, reduces RSV-induced p65Ser536 phosphorylation. A549 were transfected with control (Ctrl)-, TRAF2-, TRAF3-, or TRAF6-specific siRNA oligonucleotides (Table 1). (A) siRNA-transfected A549 cells were treated and data analyzed as described in Fig. 2A. (*, P < 0.05); n.s., not significantly different; mean ± the SEM; n = 6). The data are representative of two independent experiments. (B and C) WCE obtained from siRNA-transfected cells were analyzed by immunoblotting (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-TRAF6 (B), anti-TRAF2 (C), anti-TRAF3 (C), and anti-actin antibodies. Immunoblots are representative of three independent experiments. (D) siRNA-transfected A549 cells were further cotransfected with CD40 and CD40L expressing constructs and the P2(2×)TK-pGL3 NF-κB firefly luciferase reporter constructs or with the ISG56prom-pGL3 IRF-3 firefly reporter construct, followed by poly(I:C). The pRL-null Renilla luciferase reporter construct was used as an internal control. The data were analyzed as described in Fig. 2A. (*, P < 0.05; mean ± the SEM; n = 3.)