added and incubated overnight. Next day, after washing, the bound proteins were eluted and then separated on a SDS-PAGE followed by western blotting. Reporter assay For the luciferase reporter assay, 0.25 million 293T cells/well were seeded in 24-well plates. The next day, cells were transfected with lipofectamine 2000 (Invitrogen Life Technologies). A specified amount of DNA was added into each transfection and an empty pcDNA3.1 plasmid was included when necessary (filler DNA) to keep the total amount of DNA same in each transfection. When indicated, cells were treated with various stimuli for specified time periods. Cells were harvested between 24 and 48 h after transfection and luciferase activity was measured using the luciferase assay system (Promega). To control for transfection variations, 0.05 mg Renilla luciferase plasmid whose transcription is under the control of the neutral constitutive HSV-thymidine kinase promoter (pRL-TK) was included in each transfection (internal control). For all experiments, the data presented are the mean 6 the standard deviation (SD) of duplicate or triplicate samples and were repeated at least twice. NDV-GFP virus production NDV-GFP virus was a generous gift from Dr. Chris Basler (Mount Sinai School of Medicine, NYC) and was produced in 10- days old embryonated eggs (Charles River Laboratories Interna- tional, Inc. Wilmington, MA). The inoculated eggs were incubated for two days at 37uC and the allantoic fluid containing virus was harvested, filtered and stored in 280uC until use. The use of this virus has been described elsewhere [48]. NDV infection assay 0.5 million A549 cells were seeded in 12 well plates twelve hours prior to stimulation. Cells were stimulated in the presence of poly References 1. Medzhitov R (2001) Toll-like receptors and innate immunity. Nat Rev Immunol 1: 135–145. 2. Modlin RL (2002) Mammalian toll-like receptors. Ann Allergy Asthma Immunol 88: 543–547; quiz 548–550, 583. 3. Akira S, Takeda K (2004) Functions of toll-like receptors: lessons from KO mice. C R Biol 327: 581–589. 4. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, et al. (2005) IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol 6: 981–988. 5. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, et al. (2005) Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437: 1167–1172. 6. Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122: 669–682. 7. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, et al. (2005) VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell 19: 727–740. 8. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, et al. (2005) Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci U S A 102: 2992–2997. 9. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ (2005) Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A 102: 17717–17722. 10. Foy E, Li K, Wang C, Sumpter R, Jr., Ikeda M, et al. (2003) Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300: 1145–1148. 11. Welchman RL, Gordon C, Mayer RJ (2005) Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol 6: 599–609. 12. Chen ZJ (2005) Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol 7: 758–765. 13. Chuang TH, Ulevitch RJ (2004) Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors. Nat Immunol 5: 495–502. 14. Fearns C, Pan Q, Mathison JC, Chuang TH (2006) Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding. J Biol Chem 281: 34592–34600. PLIC-1 Inhibits TLR3 Activation I:C (100 mg/ml) for 24 hours. Cells were washed with PBS and 20 ml (MOI 1) of NDV-GFP virus was added in 180 ml of PBS (with Ca and Mg) and left at 37uC for 1 hour. Cells were then washed with PBS and incubated in fresh F12K media followed by fixation and imaging. GFP signal started to appear after 12 hours but peaked at 24 hours post-infection. DAPI was added to stain nuclei. Immunofluorescence staining and Confocal Microscopy Transfected Huh7.5.1 cells were fixed in 2% paraformaldehyde (15 min, RT), permeablized with 0.1% (v/v) Triton X-100 in PBS three times (PBST; 10 min, RT), and blocked in 0.3% (w/v) bovine serum albumin (BSA) in PBS (45 minutes, RT), and subsequently incubated in PBS (1 h, RT) with anti-Flag M2 antibody (1:500). Samples were washed in PBS and subsequently incubated with Alexa 568 secondary antibodies diluted in PBS (1:500). Nucleus was stained with Draq5 (Biostatus, United Kingdom). Samples were mounted on a slide using a homemade Gelvatol mounting medium. Images were captured on a Carl Zeiss Meta LSM 510 confocal microscope (606 Plan-Neo/1.3 Oil) and edited by Photoshop (Adobe). Acknowledgments We are grateful to Drs. T. Taniguchi, S. Smale, P. Godowski, T. Chuang, J. Pomerantz, M. Boldin, P. Howley, E. Brown, D. Golenbock, S. Saumendra, J. Chen, D. Hackam, K. Li, C. Coyne, and F. Chisari for providing plasmids and the cell lines. Author Contributions Conceived and designed the experiments: NB TW. Performed the experiments: NB SL TR SEA WL TW. Analyzed the data: NB SL TW. Contributed reagents/materials/analysis tools: TF. Wrote the paper: NB TW. 15. Nakhaei P, Mesplede T, Solis M, Sun Q, Zhao T, et al. (2009) The E3 ubiquitin ligase Triad3A negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation. PLoS Pathog 5: e1000650. 16. Bedford FK, Kittler JT, Muller E, Thomas P, Uren JM, et al. (2001) GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin- like protein Plic-1. Nat Neurosci 4: 908–916. 17. Ross CA, Pickart CM (2004) The ubiquitin-proteasome pathway in Parkinson’s disease and other neurodegenerative diseases. Trends Cell Biol 14: 703–711. 18. Kleijnen MF, Shih AH, Zhou P, Kumar S, Soccio RE, et al. (2000) The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome. Mol Cell 6: 409–419. 19. Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr. (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388: 394–397. 20. Matsumoto M, Kikkawa S, Kohase M, Miyake K, Seya T (2002) Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double- stranded RNA-mediated signaling. Biochem Biophys Res Commun 293: 1364–1369. 21. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, et al. (2003) Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301: 640–643. 22. Barton GM, Medzhitov R (2002) Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A 99: 14943–14945. 23. Heir R, Ablasou C, Dumontier E, Elliott M, Fagotto-Kaufmann C, et al. (2006) The UBL domain of PLIC-1 regulates aggresome formation. EMBO Rep 7: 1252–1258. 24. N’Diaye EN, Kajihara KK, Hsieh I, Morisaki H, Debnath J, et al. (2009) PLIC proteins or ubiquilins regulate autophagy-dependent cell survival during nutrient starvation. EMBO Rep 10: 173–179. 25. Funami K, Sasai M, Ohba Y, Oshiumi H, Seya T, et al. (2007) Spatiotemporal mobilization of Toll/IL-1 receptor domain-containing adaptor molecule-1 in response to dsRNA. J Immunol 179: 6867–6872. 26. Li K, Chen Z, Kato N, Gale M, Jr., Lemon SM (2005) Distinct poly(I-C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes. J Biol Chem 280: 16739–16747. PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e21153