amplification product was produced. Quantification of changes in gene expression was using the DDCt method using uninfected cells as a calibrator [1]. Electrophoretic Mobility Shift Assay (EMSA) A total of 35 mg whole cell extracts (WCEs) were incubated in DNA-binding buffer containing 5% glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM DTT, 5 mM MgCl2, 0.5 mM EDTA, 1 mg of poly (dA-dT), and 100,000 cpm of 32P-labeled double-stranded oligonucleotide containing NF-kB binding sites [24] and IRF3 binding site [1] in a total volume of 25 mL. After fractionation in TBE acrylamide, gels were dried and exposed to BioMax film (Kodak) for autoradiography. Native PAGE for IRF-3 Dimer Formation 50 mg protein was fractionated by 7% native acrylamide gel in running buffer containing 1% sodium deoxycholate (Sigma Aldrich) as described [25]. After electrophoresis, proteins were transferred and analyzed by Western Immunoblot. Co-Immunoprecipitation and Western Immunoblot Whole cell extracts (WCEs) were prepared using modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxy- cholate, 1% IGEPAL CA-630, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, and 1 mg/ml each of aprotinin, leupeptin, and pepstatin). WCEs were pre-cleared with protein A-Sepharose 4B (Sigma) for 10 min at 4uC and immunoprecipitation was conducted for 2 hours at 4uC with primary Ab. Immune complexes were then precipitated by adding 50 mL of protein A-Sepharose beads (50% slurry) and incubated for 1 h at 4uC. Beads were washed three times with cold TB buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl [pH 7.4], 0.05% IGEPAL CA-630), and immune complexes were fractionated by 10% SDS- polyacrylamide gel electrophoresis and transferred to a polyviny- lidene difluoride membrane by electroblotting. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline–0.1% Tween and probed with the indicated primary Ab. Membranes were washed and incubated with IRDye 700-conjugated anti- mouse Ab or IRDye 800-conjugated anti-rabbit Ab (Rockland, Inc.). Finally, the membranes were washed three times with TBS- T and imaged by an Odyssey infrared scanner. Sources of primary Ab were: anti-Flag M2 mAb (Stratagene), rabbit anti-IRF3 polyclonal Ab (Santa Cruz), anti-Myc mAb (Santa Cruz), anti- STAT1 polyclonal Ab(Santa Cruz) and anti-phospho-STAT1 (Cell Signaling). Confocal Immunofluorescence Microscopy Transfected cells were plated on cover glasses pretreated with rat tail collagen (Roche Applied Sciences). After indicated stimulation, the cells were fixed with 4% paraformaldehyde in PBS and incubated with 0.1 M ammonium chloride (10 min). Cells were permeabilized with 0.5% Triton-100, followed by incubation in blocking buffer (5% goat serum, 0.1% triton X-100, 0.05% NaN3, and 1% BSA) and incubated with Anti-Rel A Ab (c-20, sc-372, Santa Cruz) in incubation buffer (0.1% triton X-100, 0.05% NaN3, and 2% BSA) overnight at 4uC. After washing, cells were stained with Alexa Fluor 555-conjugated Goat anti-rabbit IgG (Invitrogen) in incubation buffer for 1 h. After removing secondary antibody, the cells were fixed and counterstained with DRAQ5 (2 mM). The cells were visualized by Zeiss fluorescence LSM510 confocal microscope at 63X magnification. IRF3-NF-kB Pathway Utilization Results Selective IKKc Expression in Response to ssRNA Infection Previously we found that full length IKKc-WT and the alternatively spliced IKKcD transcripts were expressed at 2:1 ratios in uninfected A549 cells [18]. To determine whether IKKc expression is affected by ssRNA virus infection, selective QRT- PCR assays were designed to measure total IKKc isoform expression. Total IKKc was quantified using primer pairs that selectively amplified the region corresponding to Exons 2–3; IKKc-WT was quantified using primer pairs that selectively amplified the Exon 4-5 boundary; and IKKcD was quantified using primers that selectively amplified the Exon 4–6 boundary (Supplementary Table SI online). A549 cells were infected with sucrose cushion purified RSV, and IKKc transcripts quantitated. Strikingly, relative to uninfected cells, total IKKc transcripts were markedly induced 55- fold 6 h after RSV infection and returned to baseline 16 h later (Figure 1A, top panel). Similarly, full length IKKc-WT was transiently induced 80-fold 6 h after RSV infection (Figure 1A, middle panel). By contrast, the IKKcD isoforms was only weakly (7-fold) induced by RSV infection at the same time point (Figure 1A, bottom panel). Together these data indicate that the differential expression of IKKc splice forms is regulated by ssRNA infection. The Replication of RNA Viruses Is Increased in IKKcD Reconstituted MEFs To selectively compare the function of IKKc and IKKcD in response to RNA viruses, IKKc2/2 mouse embryonic fibroblasts (MEFs) were transfected with full length IKKc WT or IKKcD expression vectors and isoform expression quantified by Western immunoblot (Figure 1B). At equivalent amounts of expression vector, IKKcD expressed at a 2-fold higher level than did IKKc-WT, and was not affected by RSV infection. To determine if IKKc isoform expression affects RSV replication, the expression of RSV proteins were detected in Western immunoblot using a pan-anti-RSV Ab. As expected from their inability to produce type I IFN, RSV replicated to high levels in the IKKc2/2 cells. In cells expressing IKKc-WT, the level of RSV replication was significantly reduced (Figure 1C), consistent with the robust IFN production in RSV infected cells and the actions of type I IFN to restrict RSV replication [26]. By contrast, despite the findings that ectopic IKKcD had a slightly higher expression level than that of IKKc-WT, there was a significant increase of RSV proteins produced 16 h after RSV infection (Figure 1C, compare G, N and M protein expression). Quantification of the RSV N protein by near-infrared scanning (LiCOR Odyssey) showed that the normalized abundance of N was 53 arbitrary units (AU) in IKKc2/2 MEFs, 24 AU in IKKc-WT expressing cells and 46 AU in IKKcD expressing cells. Similar findings were seen for RSV G and M proteins. To confirm this result, multinucleated cell (MNC) formation, a consequence of RSV Fusion protein expression, was measured in IKKc2/2 MEFs expressing either EGFP, EGFP-IKKc-WT or EGFP-IKKcD [18]. MNCs were quantified by scoring 100 EGFP- positive cells in 5 randomly selected images by an observer blinded to the experimental condition. Twenty MNCs were observed in RSV-infected empty vector transfectants, whereas 9 were observed in EGFP-IKKc-WT and 18 in EGFP-IKKcD transfectants. The reduction in MNC formation in IKKc-WT transfectants is highly significant compared to empty vector transfectants (p,0.01, x2 statistic ), whereas the number of MNCs in IKKcD was not different from empty vector. These data indicate that IKKcD is more permissive for RSV replication than IKKc-WT. Similar findings were produced in IKKc2/2 cells stably expressing IKKc-WT, IKKcD or empty vector (Supplementary Figure S1 online). PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e8079