ISGylation machinery in the presence or absence of USP18. Rec IFN a2b was a gift of D. Gewert (Wellcome, UK) and IFN b was from Biogen Idec (Boston, MA). Mutants IFN a2-HEQ and IFN a2-a8tail were described in [6,42]. IFN a1, a8 and a21 were produced as in [43]. IFN v was from G.R. Adolf (Bender, Wien). All IFNs were purified to homogeneity. Hyper-IL-6, chimeric fusion of human IL-6 and IL-6Ra, was a gift of Merck Serono S.A. Human IFN c was from PBL, Biochemical Laboratories and IFN l1 from Peprotech. Luciferase reporter assay To measure luciferase activity, cells were plated in 96-well plate and treated in triplicate for 6 hr with 9 dilutions of IFN in a concentration range covering 2.4 log. Cells were lysed and luciferase activity was quantified in a luminometer (LB960 Berthold). Non-linear regression fits and determination of EC50 were done using Prism 5 (GraphPad software). Quantitative real-time PCR Cells were treated with IFN for 4 hr. Total RNA was purified with RNeasy columns (Qiagen). Reverse transcriptions were primed with random primers and performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quanti- tative real-time PCR (qRT-PCR) was performed using the TaqMan gene expression assay technology (Applied Biosystems) for USP18 (catalog no. Hs00276441). Each sample was run in triplicate, normalized to the 18S RNA amplification level in the same sample, and calculated relative to the expression of the target gene in unstimulated cells. For measuring OAS 69K mRNA, qRT- PCR assays were performed as in [26]. Quantification data are presented as the 95% confidence limits of ratio to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level. Protein analysis Cells were processed as in [44]. Polyclonal antibodies (Abs) used were: anti-phospho-Tyr1054/Tyr1055 Tyk2 (Calbiochem); anti- phospho-Tyr689 Stat2; anti-phospho-Tyr701 Stat1, anti-phospho- Tyr705 Stat3, and anti-USP18 (a gift from D.E. Zhang, The Scripps Research Institute, La Jolla, CA), anti-Jak1 (UBI, Lake Placid, NY), anti-Jak1-phospho-YY1022/23 (Biosource, CA) and anti-pan Akt (Cell Signaling Technology, Beverly, MA). Mouse mAbs were anti-Tyk2 T10-2 (Hybridolab, Institut Pasteur, France); anti-phosphotyrosine 4G10 (UBI, Lake Placid, NY) and anti-ISG15 clone 2.1 (a gift from E.C Borden, Cleveland Clinic, Cleveland, Ohio). Signal was revealed with the ECL enhanced chemiluminescence Western blotting reagent (Pierce) or the more sensitive Western Lightning Chemiluminescence Reagent Plus (PerkinElmer). Signals were acquired and quantified with a Kodak Image Station 440 cf. For flow cytometric analyses we used mAbs anti-IFNAR1 AA3 (BiogenIdec, Boston) and anti-IFNAR2 CD118 References 1. Borden EC, Sen GC, Uze G, Silverman RH, Ransohoff RM, et al. (2007) Interferons at age 50: past, current and future impact on biomedicine. Nat Rev Drug Discov 6: 975–990. 2. Sommereyns C, Paul S, Staeheli P, Michiels T (2008) IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo. PLoS Pathog 4: e1000017. 3. Ferrantini M, Capone I, Belardelli F (2008) Dendritic cells and cytokines in immune rejection of cancer. Cytokine Growth Factor Rev 19: 93–107. 4. Uze G, Schreiber G, Piehler J, Pellegrini S (2007) The receptor of the type I interferon family. Curr Top Microbiol Immunol 316: 71–95. 5. Moraga I, Harari D, Schreiber G, Uze G, Pellegrini S (2009) Receptor density is key to the alpha2/beta interferon differential activities. Mol Cell Biol 29: 4778–4787. 6. Jaitin DA, Roisman LC, Jaks E, Gavutis M, Piehler J, et al. (2006) Inquiring into the differential action of interferons (IFNs): an IFN-alpha2 mutant with Differential Desensitization to Type I Interferons (P,BL, Piscataway, NJ) or D5 (BiogenIdec, Boston) as in [44]. Samples were analysed with Becton Dickinson FACScan or Canto flow cytometers. IFN binding assays IFN a2 and IFN a2-HEQ (30 mg) were labelled with Iodine125 (PerkinElmer, NEZ033A) by using a modified chloramine T method [45]. The labelled IFN preparations were titrated using a luciferase reporter assay relative to IFN a2 and IFN a2-HEQ references of known molar concentrations. The actual incorpora- tions and monomer concentrations were as follows: a2: 75 nM and 54 Bq/fmol; a2-HEQ: 25 nM and 87 Bq/fmol. For binding assays (Fig. 3C and D), na¨ıve and 8 hr-primed HLLR1-1.4 cells were seeded on 6-well plates (86105 cells/well) and 16 hr later incubated for 1 hr at 37uC with different concentrations of either 125I-IFN a2 or 125I-IFN a2-HEQ only or in the presence of a 100 fold excess of unlabeled cold IFN a2- HEQ competitor. Cells were washed three times in DMEM and 5% serum to eliminate unbound IFN, trypsinized, and counted for 125I using a c counter (Berthold). For binding assays on clones (Fig. 6B and C), cells were seeded on 6-well plates (86105 cells/ well) and treated as above. USP18 silencing USP18 ON-TARGETplus SMARTpool and a control siRNA (ON-TARGETplus non-targeting pool) were from Dharmacon. Cells were transfected with 25 nM of siRNA using Lipofectamine RNAi max reagent (Invitrogen), according to manufacturer’s instructions. Twenty-four hr later, cells were either left untreated or primed, washed and challenged with IFN a2 or IFN b for 30 min to measure activation of Stats (scheme in Fig. 1A). The siRNAs constituting the USP18 ON-TARGETplus SMARTpool were also tested individually. Acknowledgments This work is dedicated to the memory of Knud E. Mogensen. We would like to thank D.E. Zhang, E.C Borden, D. Baker, Y. Wu and M. Albert, for providing reagents and cells; I. Moraga, J. Ragimbeau and V. Libri for insightful advice and technical help; T. Rose and P. Casanova for assistance in ligand iodination; P. Maurel for support in experiments on hepatocytes, V. Mallet and all members of the laboratories for helpful discussions. We thank E. Bianchi, A. Blangy, M. Arpin, F. Michel and I. Moraga for critical reading of the manuscript. Author Contributions Conceived and designed the experiments: VF-N SP GU. Performed the experiments: VF-N GMdFA BP-B DM LP-G. Analyzed the data: VF-N SP GU. Contributed reagents/materials/analysis tools: JP. Wrote the paper: SP GU. enhanced affinity to IFNAR1 is functionally similar to IFN-beta. Mol Cell Biol 26: 1888–1897. 7. Kalie E, Jaitin DA, Podoplelova Y, Piehler J, Schreiber G (2008) The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities. J Biol Chem 283: 32925–32936. 8. Coelho LF, Magno de Freitas Almeida G, Mennechet FJ, Blangy A, Uze G (2005) Interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine CXCL11 expression. Proc Natl Acad Sci U S A 102: 11917–11922. 9. Coccia EM, Uze G, Pellegrini S (2006) Negative regulation of type I interferon signaling: facts and mechanisms. Cell Mol Biol (Noisy-le-grand) 52: 77–87. 10. Sarasin-Filipowicz M, Wang X, Yan M, Duong FH, Poli V, et al. (2009) Alpha interferon induces long-lasting refractoriness of JAK-STAT signaling in the mouse liver through induction of USP18/UBP43. Mol Cell Biol 29: 4841–4851. PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22200