IRF3-NF-kB Pathway Utilization Figure 6. The IKKcD:IKKc-WT ratio affects viral inducible type I IFN expression. (A) IKKc2/2 MEFs were transfected with mixtures of eukaryotic expression vectors encoding IKKc-WT, or IKKcD keeping the total IKKc expression plasmid constant as indicated. Shown is quantitation of the Western immunoblot for the b-actin normalized expression for each isoform. (B) Effect on IFN expression. Transfectants were RSV infected for 0 or 16 h. Total cellular RNA was assayed by Q-RT-PCR for the expression of IFNb, IFNa1, IRF7 or IFNa4 as indicated. Shown is X6SD of the fold mRNA induction relative to uninfected IKKc-WT transfected cells. (C) Effect on NF-kB dependent gene expression. RNA was assayed by QRT-PCR for Grob and A20 expression as indicated. Results were repeated three times with similar results. doi:10.1371/journal.pone.0008079.g006 IKK-a, -b or -e was co-transfected with Myc tagged IKKc-WT or IKKcD and subjected to nondenaturing coimmunoprecipitation. To demonstrate the essential role of TANK for recruiting IKKe, V5-labeled TANK was also co-transfected with IKKe. After IKKc isoforms were precipitated using anti-Myc Ab, the association of respective IKK was detected by anti-Flag Ab. Consistent with our previous work, IKKc-WT associates with IKK-a and IKK-b [18]. IKKe did not bind to IKKc-WT in the absence of TANK, but in cells cotransfected with TANK, IKKe could bind (Figure 8C, left panel). Conversely, although IKKcD bound IKKa and IKKb, it did not recruit IKKe, even in the presence of TANK (Figure 8C, right panel). Based on these data, we conclude that IKKcD is PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e8079