IRF3-NF-kB Pathway Utilization Figure 1. Enhanced viral protein expression in IKKcD reconstituted cells. (A) QRT-PCR of IKKc isoforms in response to RSV infection. A549 cells were RSV infected (M.O.I. = 1) for indicated times, after which total RNA was extracted and assayed by QRT-PCR for IKKc. Top panel, QRT-PCR for total IKKc isoforms (using primers spanning Exon 2–3). Middle panel, QRT-PCR for IKKc-WT isoform (using primers spanning the Exon 4–6 junction). Bottom panel, QRT-PCR for IKKcD isoform (using primers spanning the Exon 4–5 junction). Shown is fold change relative to uninfected cells. Data was reproduced twice, with similar results. (B) IKKc2/2-deficient MEFs were reconstituted with empty vector (IKKc2/2), IKKc-WT or IKKcD and RSV infected (MOI = 1) for 0 or 16 h. 100 mc whole cell extracts (WCEs) were assayed by Western immunoblot using anti-FLAG Ab. The locations of the two isoforms are shown. Bottom blot, the blot was reprobed with b-Actin Ab as a loading control (the FLAG bands are still visible). (C) Expression of RSV proteins were detected in WCEs using anti-pan RSV Ab (upper). b-Actin staining is used as a loading control (lower). (D) IKKc2/2-deficient MEFs were transfected with expression vectors encoding EGFP, IKKg-WT-EGFP, or IKKgD-EGFP as indicated. Cells were then mock or RSV infected (M.O.I. = 1) for 24 h. Cells were fixed, stained with DRAQ5 (2 mM, Biostatus UK) and imaged by fluorescence microscopy in an LSM510 confocal microscope (magnification of 63X). Representative multinucleated cells contained within a single plasma membrane are indicated by white arrows. (E) IKKc2/2-deficient MEFs reconstituted with empty vector (IKKc2/2), IKKc-WT or IKKcD were Sendai virus infected (MOI = 1) for 0, 12, 24 h. Protein expression was detected in WCEs using anti- Sendai Virus Ab (upper). b-Actin staining is used as a loading control (lower). doi:10.1371/journal.pone.0008079.g001 We also investigated Sendai virus (SeV) replication in IKKc-WT and IKKcD reconstituted IKKc2/2 MEFs. Both at 12- and 24 h after infection, a significant increase of Sendai viral protein was observed in IKKc2/2 and IKKcD reconstituted MEFs, as compared to those reconstituted with IKKc-WT (Figure 1E). Quantification of the normalized protein abundance is shown in the PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e8079