Figure 8. IKKcD does not interact with TANK?IKKe. (A) Myc epitope-tagged IKKc or IKKcD was co-transfected with Flag-MAVS into HEK293 cells. 48 h later, 500 mg WCEs were collected and incubated with a control mouse IgG or anti-Myc Ab as indicated. Nondenaturing co-immunoprecipitation (IP) was performed using anti-Myc Ab; MAVS association was detected by Western immunoblot (IB) with anti-Flag Ab (top panel). Presence of Myc-IKKc and Myc-IKKcD in the immunopre- cipitates were detected by IB using anti-Myc Ab. Specific bands indicated by asterisks. ns, nonspecific band. (B) Myc epitope-tagged IKKc or IKKcD was transfected into HEK293 cells with V5 labeled TANK. 48 h later, WCEs were immunoprecipitated with control (C) or anti-Myc Ab (Myc). TANK association was determined by IB using anti-V5 Ab. TANK is indicated by the black arrowhead. Bottom, the presence of Myc-IKKc or IKKcD in immunoprecipitates were confirmed using anti- Myc Ab. Specific bands indicated by asterisks. (C) Flag epitoped-tagged IKKa, IKKb or IKKe was co-transfected with Myc-IKKc or Myc-IKKcD. IKKe was transfected in the absence or presence of TANK. 48 h later, WCEs were collected and subjected to nondenaturing co-immunoprecipita- tion using anti-Myc Ab. IKKa, IKKb or IKKe association was detected by anti-Flag antibody (top panel). The presence of Myc-IKKc, Myc-IKKcD and V5-TANK was demonstrated in the immune complexes by Western immunoblot. Expression of respective proteins was also confirmed in whole cell lysates using Western immunoblot (Lysate, bottom three panels). Location of specific bands are indicated at right. doi:10.1371/journal.pone.0008079.g008 MAVS complex with an isoform deficient in recruiting TANK- IKKe. Moreover, in cells naturally expressing high levels of endogenous IKKcD to IKKc-WT ratios are defective in viral inducible IRF3 activation but respond via cytokine induced NF- kB activators. Strikingly, IKKc-WT mRNA expression is highly inducible by ssRNA infection relative to IKKcD, suggesting that the cellular ratios of the two isoforms are dynamic. The mechanisms for this induction, and consequences in signaling will require further exploration. Together these findings indicate that relative endogenous expression of IKKcD isoforms may produce IKK complexes that differentially couple to distinct upstream IRF3-NF-kB Pathway Utilization Figure 9. IKKcD competes with IKKc-WT for MAVS binding. IKKc2/2 MEFs were cotransfected with eukaryotic expression vectors encoding Myc-IKKc-WT and FLAG-MAVS in the absence or presence of Myc-IKKcD. 36 h later, nondenaturing coimmunoprecipation experi- ments were conducted using anti-FLAG. Association of IKKc isoforms were detected in the Western immunoblot using anti-Myc Ab. doi:10.1371/journal.pone.0008079.g009 signals, affording heterogeneity in cellular responses to otherwise similar activating stimuli (Figure 10). IKKc is encoded by a 10-exon-containing gene located at chromosome Xq28 [39]. Mutations in the IKKc gene including different truncations of the IKKc protein have been linked to the human syndromes of incontinentia pigmenti and anhidrotic ectodermal dysplasia associated with immunodeficiency [40]. Although human IKKc transcripts containing an alternatively spliced first (noncoding) exon have been deposited in GenBank (AI24572, AF091453), these alternatively spliced transcripts encode wild type IKKc [39]. IKKcD is the only alternative splice form known that affects the IKKc coding region, and is caused by occlusion of exon 5. Using both 2D gel electrophoresis and a reverse transcription-PCR assay that distinguished the two isoforms, we found that IKKcD is widely expressed in normal human tissues in various relative ratios with fully spliced IKKc- WT [18]. For example in normal breast and cervical tissue, IKKcD is the predominant isoform detected, whereas in normal liver and lung IKKc-WT is predominant (HeLa S3, derived from a human cervical tumor maintains this IKKcD predominance). The findings that IKKcD expression reduces IRF3 signaling and type I IFN response in response to ssRNA virus infection may yield new insight for tissue-selective differences in anti-viral responses and tissue tropism in RNA virus infections [41]. We note that viral replication in IKKcD-expressing MEFs is enhanced relative to those cells expressing IKKc-WT, but not to the degree seen in IKKc2/2 MEFs (Figure 1C). In these experiments, viral inducible expression of the major type I IFNs, IFN-b, -a4 and -a1, is nearly absent and indistinguishable from that in IKKc2/2 cells. That this degree of inhibition is biologically significant is demonstrated by the lack of detectable phospho-STAT formation or STAT autoregulation (Figure 2). It is surprising, then, that virus does not replicate in IKKcD2expressing cells to a similar degree as that seen in IKKc2/2 cells. One interpretation of these findings is that IKKcD expressing cells, residual NF-kB activity may play a PLoS ONE | www.plosone.org 12 November 2009 | Volume 4 | Issue 11 | e8079