[Roche, Penzberg, Germany]) and incubated on ice for 30 minutes. Western blotting The cell lysates (25–40 mg total protein or 15 mg nuclear protein per lane) were submitted to SDS- PAGE (on 12% gels), transferred to polyvinylidenediflouride membranes for Western Blot analysis. After blocking with 5% fat-free dried milk or BSA dissolved in TBS-T for 1 h at room temperature, membranes were incubated over night with antibodies raised against phosphorylated SAPK/ JNK, p38, ERK, IkBa, IRF3 and total IRF3, SOCS2(Cell Signaling Technology, Beverly, MA) and p65NF-kB (BD Biosci- ences, Heidelberg, Germany) respectively according to the manufacturer’s instructions. Binding of these primary antibodies was visualized with goat anti-rabbit/anti-mouse immunoglobulin coupled to horseradish peroxidase (Santa Cruz biotechnology, CA, USA). After stripping, the membranes were incubated and re- probed for new antibodies. Measurement of total ERK, IRF3 and b-actin protein served as loading controls. References 1. Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124: 783–801. 2. Mellman I, Steinman RM (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106: 255–258. 3. Steinman RM, Hawiger D, Nussenzweig MC (2003) Tolerogenic dendritic cells. Annu Rev Immunol 21: 685–711. 4. Moser M, Murphy KM (2000) Dendritic cell regulation of TH1–TH2 development. Nat Immunol 1: 199–205. 5. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392: 245–252. 6. Schnurr M, Then F, Galambos P, Scholz C, Siegmund B, et al. (2000) Extracellular ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells. J Immunol 165: 4704–4709. 7. Visintin A, Mazzoni A, Spitzer JH, Wyllie DH, Dower SK, et al. (2001) Regulation of Toll-like receptors in human monocytes and dendritic cells. J Immunol 166: 249–255. 8. Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635–700. 9. Gruber A, Mancek M, Wagner H, Kirschning CJ, Jerala R (2004) Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition. J Biol Chem 279: 28475–28482. 10. O’Neill LA (2008) When signaling pathways collide: positive and negative regulation of toll-like receptor signal transduction. Immunity 29: 12–20. 11. Hertzog PJ, O’Neill LA, Hamilton JA (2003) The interferon in TLR signaling: more than just antiviral. Trends Immunol 24: 534–539. 12. 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. 13. Randolph GJ, Inaba K, Robbiani DF, Steinman RM, Muller WA (1999) Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity 11: 753–761. 14. Dauer M, Obermaier B, Herten J, Haerle C, Pohl K, et al. (2003) Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors. J Immunol 170: 4069–4076. 15. Sallusto F, Lanzavecchia A (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179: 1109–1118. 16. Zhou LJ, Tedder TF (1996) CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci U S A 93: 2588–2592. 17. Piemonti L, Bernasconi S, Luini W, Trobonjaca Z, Minty A, et al. (1995) IL-13 supports differentiation of dendritic cells from circulating precursors in concert with GM-CSF. Eur Cytokine Netw 6: 245–252. 18. Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, et al. (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A 95: 114–119. 19. Kubo M, Hanada T, Yoshimura A (2003) Suppressors of cytokine signaling and immunity. Nat Immunol 4: 1169–1176. 20. Alexander WS, Hilton DJ (2004) The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol 22: 503–529. 21. Kinjyo I, Hanada T, Inagaki-Ohara K, Mori H, Aki D, et al. (2002) SOCS1/ JAB is a negative regulator of LPS-induced macrophage activation. Immunity 17: 583–591. SOCS2 and DC Maturation Statistical analysis Statistical comparisons between groups were made by analysis of variance followed by T-test or Fisher’s post-hoc test; p-values ,0.05 were considered significant. Supporting Information Table S1 Oligonucleotide sequences used for siRNA Found at: doi:10.1371/journal.pone.0007178.s001 (0.03 DOC) MB Table S2 Oligonucleotide sequences used for real-time PCR Found at: doi:10.1371/journal.pone.0007178.s002 (0.05 MB DOC) Author Contributions Conceived and designed the experiments: JH OW ACW GN. Performed the experiments: JH AFM. Analyzed the data: JH OW AFM ACW. Contributed reagents/materials/analysis tools: OW GN. Wrote the paper: JH OW ACW GN. 22. Lang T, Mansell A (2007) The negative regulation of Toll-like receptor and associated pathways. Immunol Cell Biol 85: 425–434. 23. Yoshimura A, Naka T, Kubo M (2007) SOCS proteins, cytokine signalling and immune regulation. Nat Rev Immunol 7: 454–465. 24. Rico-Bautista E, Flores-Morales A, Fernandez-Perez L (2006) Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions. Cytokine Growth Factor Rev 17: 431–439. 25. Jackson SH, Yu CR, Mahdi RM, Ebong S, Egwuagu CE (2004) Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling. J Immunol 172: 2307–2315. 26. Baetz A, Frey M, Heeg K, Dalpke AH (2004) Suppressor of cytokine signaling (SOCS) proteins indirectly regulate toll-like receptor signaling in innate immune cells. J Biol Chem 279: 54708–54715. 27. Machado FS, Johndrow JE, Esper L, Dias A, Bafica A, et al. (2006) Anti- inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent. Nat Med 12: 330–334. 28. Strengell M, Lehtonen A, Matikainen S, Julkunen I (2006) IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells. J Leukoc Biol 79: 1279–1285. 29. Bottcher I, Bellinghausen I, Konig B, Knop J, Saloga J (2008) Different regulation of T helper 1- and T helper 2-promoting cytokine signalling factors in human dendritic cells after exposure to protein versus contact allergens. Immunology 123: 139–144. 30. Akira S, Hoshino K, Kaisho T (2000) The role of Toll-like receptors and MyD88 in innate immune responses. J Endotoxin Res 6: 383–387. 31. Martin MU, Wesche H (2002) Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family. Biochim Biophys Acta 1592: 265–280. 32. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, et al. (2001) Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 167: 5887–5894. 33. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, et al. (2002) TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol 3: 392–398. 34. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, et al. (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18: 767–811. 35. Carmody RJ, Chen YH (2007) Nuclear factor-kappaB: activation and regulation during toll-like receptor signaling. Cell Mol Immunol 4: 31–41. 36. Doyle S, Vaidya S, O’Connell R, Dadgostar H, Dempsey P, et al. (2002) IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 17: 251–263. 37. Nakahara T, Moroi Y, Uchi H, Furue M (2006) Differential role of MAPK signaling in human dendritic cell maturation and Th1/Th2 engagement. J Dermatol Sci 42: 1–11. 38. Flores-Morales A, Greenhalgh CJ, Norstedt G, Rico-Bautista E (2006) Negative regulation of growth hormone receptor signaling. Mol Endocrinol 20: 241–253. 39. Bullock AN, Debreczeni JE, Edwards AM, Sundstrom M, Knapp S (2006) Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc Natl Acad Sci U S A 103: 7637–7642. 40. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, et al. (2002) The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27: 235–241. PLoS ONE | www.plosone.org 7 September 2009 | Volume 4 | Issue 9 | e7178