Early Growth Response-1 Regulates Lipopolysaccharide-induced Suppressor of Cytokine Signaling-1 Transcription
2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês
10.1074/jbc.m408938200
ISSN1083-351X
AutoresJustin Mostecki, Brian M. Showalter, Paul B. Rothman,
Tópico(s)Chemokine receptors and signaling
ResumoSuppressor of cytokine signaling (SOCS)-1 is a critical regulator of lipopolysaccharide (LPS) tolerance and LPS-induced cytokine production. The mechanisms regulating the transcription of SOCS-1 in response to LPS are not entirely understood. Functional analysis of the SOCS-1 promoter demonstrates that early growth response-1 (Egr-1) is an important transcriptional regulator of SOCS-1. Two Egr-1 binding sites are present within the SOCS-1 promoter as shown by EMSA and supershift analysis. Further, mutation of the Egr-1 binding sites significantly reduces both the basal and LPS-induced transcriptional activity of the promoter. Chromatin immunoprecipitation experiments confirm LPS-induced binding of Egr-1 to the SOCS-1 promoter in vivo. Additionally, Egr-1–/– macrophages show reduced levels of LPS-induced SOCS-1 expression in comparison with macrophages derived from Egr-1+/+ littermate controls. These results demonstrate an important role for Egr-1 in regulating both the basal and LPS-induced activity of the SOCS-1 promoter. Suppressor of cytokine signaling (SOCS)-1 is a critical regulator of lipopolysaccharide (LPS) tolerance and LPS-induced cytokine production. The mechanisms regulating the transcription of SOCS-1 in response to LPS are not entirely understood. Functional analysis of the SOCS-1 promoter demonstrates that early growth response-1 (Egr-1) is an important transcriptional regulator of SOCS-1. Two Egr-1 binding sites are present within the SOCS-1 promoter as shown by EMSA and supershift analysis. Further, mutation of the Egr-1 binding sites significantly reduces both the basal and LPS-induced transcriptional activity of the promoter. Chromatin immunoprecipitation experiments confirm LPS-induced binding of Egr-1 to the SOCS-1 promoter in vivo. Additionally, Egr-1–/– macrophages show reduced levels of LPS-induced SOCS-1 expression in comparison with macrophages derived from Egr-1+/+ littermate controls. These results demonstrate an important role for Egr-1 in regulating both the basal and LPS-induced activity of the SOCS-1 promoter. The Toll-like receptor (TLR) 1The abbreviations used are: TLR, Toll-like receptor; TNF, tumor necrosis factor; LPS, lipopolysaccharide; SOCS-1, suppressor of cytokine signaling; JAK, c-Jun-activated kinase; STAT, signal transducers and activators of transcription; IFN, interferon; IRF-E, IRF binding element; Egr-1, early growth response factor-1; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; S1 and S2, site 1 and site 2, respectively.1The abbreviations used are: TLR, Toll-like receptor; TNF, tumor necrosis factor; LPS, lipopolysaccharide; SOCS-1, suppressor of cytokine signaling; JAK, c-Jun-activated kinase; STAT, signal transducers and activators of transcription; IFN, interferon; IRF-E, IRF binding element; Egr-1, early growth response factor-1; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; S1 and S2, site 1 and site 2, respectively. family is a diverse group of transmembrane receptors that recognize microbial products called pathogen-associated molecular patterns. The proper regulation of TLR signaling is important in limiting septic shock, inflammation, and various aspects of both innate and adaptive immunity. The ligation of the TLR4-MD2 complex by LPS induces the recruitment of the MyD88 and subsequent activation of both interleukin-1 receptor-associated kinase and TNF receptor-associated factor-6. Activation of these molecules leads to signaling via the NF-κB and the c-Jun N-terminal kinase mitogen-activated protein kinase pathways. Whereas a significant response to a bacterial infection is often required for protective immunity, elevated levels of cytokine protection can have deleterious effects; for example, toxic shock and systemic inflammatory syndrome. Repeated exposure to LPS induces a protective condition, termed LPS tolerance, that prevents shock following challenge with otherwise lethal levels of endotoxin. Various mechanisms have been proposed for the development of this refractory state: TLR4 down-regulation (1Nomura F. Akashi S. 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Biol. Chem. 2000; 275: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). SOCS-1 is a critical regulator of IFN-γ signaling as demonstrated by the lethality of the SOCS-1–/– neonates. While normal at birth, SOCS-1-deficient neonates display stunted growth, multiorgan disease characterized by severe lymphopenia, fatty degeneration of the liver, and macrophage infiltration of various tissues, and they die by 3 weeks of age (16Starr R. Metcalf D. Elefanty A.G. Brysha M. Willson T.A. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14395-14399Crossref PubMed Scopus (377) Google Scholar).SOCS-1 regulates various aspects of innate immunity, since it has been shown to limit LPS-induced cytokine production and be critical for the regulation of LPS tolerance (4Kinjyo I. Hanada T. Inagaki-Ohara K. Mori H. Aki D. Ohishi M. Yoshida H. Kubo M. Yoshimura A. Immunity. 2002; 17: 583-591Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 5Nakagawa R. Naka T. Tsutsui H. Fujimoto M. Kimura A. Abe T. Seki E. Sato S. Takeuchi O. Takeda K. Akira S. Yamanishi K. Kawase I. Nakanishi K. Kishimoto T. Immunity. 2002; 17: 677-687Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 17Kubo M. Hanada T. Yoshimura A. Nat. Immunol. 2003; 4: 1169-1176Crossref PubMed Scopus (537) Google Scholar). LPS and CpG DNA, which signal through the TLR-4 and TLR-9 receptors, respectively, have been shown to up-regulate SOCS-1 in macrophages (18Bode J.G. Nimmesgern A. Schmitz J. Schaper F. Schmitt M. Frisch W. Haussinger D. Heinrich P.C. Graeve L. FEBS Lett. 1999; 463: 365-370Crossref PubMed Scopus (176) Google Scholar, 19Crespo A. Filla M.B. Russell S.W. Murphy W.J. Biochem. J. 2000; 349: 99-104Crossref PubMed Scopus (84) Google Scholar, 20Dalpke A.H. Opper S. Zimmermann S. Heeg K. J. Immunol. 2001; 166: 7082-7089Crossref PubMed Scopus (205) Google Scholar). Further, both SOCS-1+/– and SOCS-1–/– mice are hyperresponsive to LPS, demonstrate reduced viability to LPS challenge, and produce elevated levels of LPS-induced proinflammatory cytokines such as interleukin-12 and TNF-α (4Kinjyo I. Hanada T. Inagaki-Ohara K. Mori H. Aki D. Ohishi M. Yoshida H. Kubo M. Yoshimura A. Immunity. 2002; 17: 583-591Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 5Nakagawa R. Naka T. Tsutsui H. Fujimoto M. Kimura A. Abe T. Seki E. Sato S. Takeuchi O. Takeda K. Akira S. Yamanishi K. Kawase I. Nakanishi K. Kishimoto T. Immunity. 2002; 17: 677-687Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Consistent with its role as a negative regulator of TLR signaling, forced expression of SOCS-1 inhibits TLR4 signaling in macrophages (4Kinjyo I. Hanada T. Inagaki-Ohara K. Mori H. Aki D. Ohishi M. Yoshida H. Kubo M. Yoshimura A. Immunity. 2002; 17: 583-591Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar) and targets p65 NF-κB for ubiquitin-mediated proteolysis (14Ryo A. Suizu F. Yoshida Y. Perrem K. Liou Y.C. Wulf G. Rottapel R. Yamaoka S. Lu K.P. Mol. Cell. 2003; 12: 1413-1426Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar).At present, the mediators for LPS induction of SOCS-1 are not entirely clear. Regulated by both transcriptional and post-transcriptional mechanisms, SOCS-1 levels are most potently up-regulated by IFN-γ in an STAT-1-dependent manner (21Saito H. Morita Y. Fujimoto M. Narazaki M. Naka T. Kishimoto T. J. Immunol. 2000; 164: 5833-5843Crossref PubMed Scopus (86) Google Scholar). The IFN-γ-inducible activity of the murine promoter is dependent upon an IRF binding element (IRF-E) (21Saito H. Morita Y. Fujimoto M. Narazaki M. Naka T. Kishimoto T. J. Immunol. 2000; 164: 5833-5843Crossref PubMed Scopus (86) Google Scholar). 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Chem. 2002; 277: 2345-2352Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).LPS stimulation activates many transcription factors downstream of the c-Jun N-terminal kinase/mitogen-activated protein kinase pathway, one of which is the zinc finger transcription factor early growth response-1 (Egr-1), also known as zif268, Krox-24, tis-8, and NGFI-A. Egr-1 is an immediate early gene that is up-regulated by a multitude of growth factors, cytokines, and environmental stresses such as hypoxia (24Yan S.F. Fujita T. Lu J. Okada K. Shan Zou Y. Mackman N. Pinsky D.J. Stern D.M. Nat. Med. 2000; 6: 1355-1361Crossref PubMed Scopus (393) Google Scholar), vascular injury (25Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar), and septic shock (26Pawlinski R. Pedersen B. Kehrle B. Aird W.C. Frank R.D. Guha M. Mackman N. Blood. 2003; 101: 3940-3947Crossref PubMed Scopus (81) Google Scholar). 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In contrast to these results, macrophage differentiation appears normal in Egr-1–/– mice (39Lee S.L. Wang Y. Milbrandt J. Mol. Cell. Biol. 1996; 16: 4566-4572Crossref PubMed Google Scholar). Further, many of the target genes of Egr-1 have additional regulatory mechanisms, because the Egr-1–/– mice demonstrate only slightly reduced levels of MCP-1, tissue factor, intercellular adhesion molecule-1, and interleukin-6 at certain time points following LPS challenge (26Pawlinski R. Pedersen B. Kehrle B. Aird W.C. Frank R.D. Guha M. Mackman N. Blood. 2003; 101: 3940-3947Crossref PubMed Scopus (81) Google Scholar).We present evidence that the LPS-induced activity of the SOCS-1 promoter is regulated by the transcription factor Egr-1. SOCS-1 expression is reduced in LPS-stimulated macrophages from Egr-1–/– versus Egr-1+/+ mice, and chromatin immunoprecipitation (ChIP) analysis indicates that the SOCS-1 promoter is bound by Egr-1 in vivo. Further, two Egr-1 binding sites are present within the SOCS-1 promoter, and mutational analysis shows that the LPS response requires these sites and an IRF-E for LPS-induced activity of the SOCS-1 promoter. Taken together, these data suggest that the SOCS-1 promoter is regulated through both Egr-1-dependent and Egr-1-independent regulatory pathways.EXPERIMENTAL PROCEDURESHuman DNA Samples—NA12547 was purchased from the Coriell Cell Repositories.Cell Lines—THP-1, a human monocytic leukemia cell line, was obtained from the American Type Culture Collection. The RAW/FPR.10 clone derived from the RAW 264.7 murine macrophage cell line was obtained from Dr. Steve Greenberg (40Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (365) Google Scholar).Luciferase Experiments—The RAW/FPR.10 cells were transfected using Lipofectamine reagent (Invitrogen). Cells were plated at a density of 3 × 105 cells/well in a 6-well plate 24 h prior to transfection. Transfection was performed following the manufacturer's protocol with 1 μg of reporter plasmid and 100 ng of Renilla luciferase control plasmid (Promega) for 4 h. Following this period, the transfection mixture was removed and replaced with media with or without 100 ng/ml LPS for 20 h. Cell extracts were subsequently prepared and assayed using the Dual Luciferase kit (Promega) as per the manufacturer's instructions. Luciferase activities were normalized to the Renilla control plasmid, and values shown are the mean of three independent experiments.Mice—The Egr-1–/– mice were provided by Dr. Shi-Fang Yan.Isolation and Differentiation of Macrophages—Bone marrow-derived macrophages were isolated and generated with an adapted method (41Lutz M.B. Kukutsch N. Ogilvie A.L. Rossner S. Koch F. Romani N. Schuler G. J. Immunol. Methods. 1999; 223: 77-92Crossref PubMed Scopus (2473) Google Scholar). Instead of granulocyte-macrophage colony-stimulating factor, the culture medium was supplemented with 10% macrophage colony-stimulating factor-containing L929 culture supernatant at day 0. At day 3, an additional 10 ml of medium containing 10% L929 supernatant was added. Day 7 adherent cells were analyzed by fluorescence-activated cell sorting and used in experiments.Probes—The sequence of the TNF-α competitor probe is 5′-AACCCTCTGCCCCCGCGATGGAG-3′.Plasmids—The IRF-2/pRSET plasmid used for the isolation of recombinant IRF-2 was provided by Dr. Kathryn Calame. Regions of the SOCS-1 promoter were cloned into the BglII and MluI sites of the pGL3 Basic Vector (Promega) using primers with the appropriate restriction sites, with the exception of the –1521/–659pGL3basic, –1160/–659pGL3basic, and –945/–659pGL3basic plasmids. The latter were generated by digestion of the –2523/–659pGL3basic plasmid with SacI, SacI + AvrII, or SacI + XhoI, respectively, followed by digestion of the overhangs with mung bean nuclease and ligation. Sequence mutations were made with the appropriate primers in a PCR-based mutagenesis protocol (Stratagene). All plasmids were confirmed by sequencing. The pRL-TK Renilla luciferase plasmid was purchased from Promega.Antibodies—The anti-IRF-1 (sc-640X), anti-IRF-2 (sc-498X), and anti-Egr-1 (sc-110X) antibodies for the supershift and ChIP experiments were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-SOCS-1 monoclonal antibody (4H1) was provided by Dr. Douglas Hilton. The anti-CD11b-phycoerythrin-conjugated and anti-CD16/CD32 (FcIII/FcII receptor) (Fc block) antibodies were purchased from Pharmingen.Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared as described previously (42Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2209) Google Scholar). Recombinant IRF-2 was purified as described in Ref. 43Merika M. Williams A.J. Chen G. Collins T. Thanos D. Mol. Cell. 1998; 1: 277-287Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar. Binding reactions were performed for 15 min with either the indicated amounts of recombinant protein or 10 μg of nuclear extracts with a final concentration of 5.0 mm HEPES (pH 7.9), 2.5 mm MgCl2, 20% glycerol, 2.5 mm dithiothreitol, 1.0 μg of dIdC and 40 μg of bovine serum albumin. Reactions were run in gels containing 6.0% acrylamide, 2.5% glycerol, and 0.25× TBE.ChIP—THP-1 cells were treated with a final concentration of 1% formaldehyde to cross-link for 10 min and subsequently stopped with the addition of glycine at a final concentration of 0.125 m for 5 min. Cells were washed twice with TBS (20 mm Tris-Cl, pH 7.4, 150 mm NaCl), counted so that 1 × 107 cells were used in each immunoprecipitation and lysed in cold SDS buffer (0.5% SDS, 100 mm NaCl, 50 mm Tris-Cl, pH 8.1, 5 mm, EDTA, pH 8.0, and 0.02% NaN3). Cells were then pelleted and resuspended in cold IP buffer (1 volume of SDS buffer plus 0.5 volume of Triton dilution buffer (100 mm Tris-Cl, pH 8.6, 100 mm NaCl, 5 mm EDTA, pH 8.0, 0.02% NaN3, and 5% Triton X-100)). The protease inhibitors aprotinin, pepstatin, and phenylmethylsulfonyl fluoride were previously added to the IP buffer. Cells were then sonicated at 4 °C and spun to remove cell debris. Extract volume was adjusted to 1.0 ml per immunoprecipitation in 1.5-ml siliconized Eppendorf tubes and precleared with 30 μl of Protein A beads (Amersham 50% slurry containing 0.2 mg/ml sonicated salmon sperm DNA and 0.5 mg/ml lipid-free bovine serum albumin) for 30 min at 4 °C. Extracts were spun to remove beads, and 25 μl of lysate was removed as a total control. Primary antibodies were then added and rotated overnight at 4 °C. 30 μl of single-stranded DNA/Protein A beads were then added the following day and rotated for an additional 2 h at 4 °C before precipitating beads and washed as follows: 2 × 1.0 ml of mixed micelle buffer (150 mm NaCl, 20 mm Tris-Cl, pH 8.1, 5 mm EDTA, pH 8.0, 0.052% (w/v) sucrose, 0.02% NaN3, 1% Triton X-100, and 0.2% SDS), 2 × 1.0 ml of buffer 500 (0.1% (w/v) deoxycholic acid, 1 mm EDTA, 50 mm HEPES, pH 7.5, 500 mm NaCl, 1% Triton X-100, and 0.02% NaN3), 2 × 1.0 ml of LiCl/detergent wash (0.5% (w/v) deoxycholic acid, 1 mm EDTA, 250 mm LiCl, 0.5% Nonidet P-40, 10 mm Tris-Cl, pH 8.0, and 0.02% NaN3), and 2 × 1.0 ml of TE (10 mm Tris-Cl, pH 8.0, and 1 mm EDTA, pH 8.0). 0.3 ml of a 1% SDS, 100 mm NaHCO3 solution was then added to both the immunoprecipitates and the total controls and incubated at 65 °C to elute complexes from beads and reverse the cross-links. Eluate was transferred to a fresh microcentrifuge tube with 0.25 ml of a proteinase K solution (30 μg of glycogen, 100 μg of proteinase K in TE, pH 7.6) and incubated at 37 °C for 2 h. 50 μl of 4 m LiCl was then added, followed by 0.5 ml of 1:1 phenol/chloroform mixture. Tubes were then shaken vigorously for 1 min and phases separated by centrifugation for 10 min at room temperature. The upper phase was transferred to a separate tube with 1.0 ml of 100% ethanol and incubated on dry ice for 15 min to precipitate DNA. DNA was centrifuged at 4 °C for 30 min, washed with 70% ethanol, recentrifuged for 5 min at 4 °C, allowed to air dry for 10 min, and resuspended in 30 μl of H2O. Input samples represent 0.05, 0.01, and 0.002% of the total DNA, whereas the PCRs of the immunoprecipitations include 10, 5, and 2.5% of the resuspended DNA. The general conditions for PCR are as follows: an initial step of 94 °C for 5 min, followed by 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s repeated for 30 cycles and 72 °C for 7 min. Each 25-μl PCR contained 1× High Fidelity PCR buffer, 2.0 mm MgSO4, 0.8 mm deoxynucleoside triphosphate, 0.8 μm each primer, and 1.25 units of HiFi Platinum TaqDNA polymerase (Invitrogen). Primers for ChIP were as follows: promoter-5′, 5′-GTCGCCAAGTCCGAAGGA-3′; promoter-3′, 5′-CCCAGCTCCACTTTTGGT-3′; 3′-control-5′, 5′-CACTAGGCAACCGGAGGA-3′; 3′-control-3′, 5′-CTTTTGAGGAGGACCTGT-3′.RESULTSRegulation of the SOCS-1 Promoter in Response to LPS—The mechanism by which LPS induces SOCS-1 transcription has not been fully elucidated. Previous evidence has suggested that the regulation is dependent upon autocrine and paracrine cytokine signaling mechanisms, whereby the treatment of macrophages with LPS induces cytokines such as IFN-α/β to up-regulate SOCS-1 expression (19Crespo A. Filla M.B. Russell S.W. Murphy W.J. Biochem. J. 2000; 349: 99-104Crossref PubMed Scopus (84) Google Scholar, 20Dalpke A.H. Opper S. Zimmermann S. Heeg K. J. Immunol. 2001; 166: 7082-7089Crossref PubMed Scopus (205) Google Scholar). The JAK-STAT-independent nature of TLR signaling suggested the possibility of an additional pathway for the regulation of SOCS-1 transcription. To determine the transcriptional elements required for regulation of SOCS-1 in response to LPS, promoter constructs were cloned using DNA isolated from a human cell line. The cloned sequence corresponds with the most common allele of human SOCS-1 (data not shown). The sequence (Fig. 1) illustrates the SOCS-1 genomic locus from the –882-position relative to the translation ATG start codon. The intron/exon structure (shown by the boxed area) is defined by the cDNA sequence of SOCS-1 (GenBank™ accession number NM_003745). Both the human and murine sequence of SOCS-1 contain two exons, with the entire coding region present within the second exon.Fig. 1Structure of the human SOCS-1 promoter region. The sequence of the first and beginning of the second exon are in the shaded regions. The translation ATG initiation site is depicted in boldface letters. The GAAA repeat IRF-E is illustrated in italic type. Four GC-boxes are underlined (GGGCGG consensus). Egr-1 site 1 and site 2 (GCGGGGGCG consensus) are shown in outline and labeled as S1 and S2, respectively. The proximal GC-box overlaps with S2. The transcription initiation site is denoted with a bent arrow, and the numbers indicate the relative position from the A(+1) of the ATG initiation site.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Various regions of the human SOCS-1 promoter were cloned upstream of a luciferase reporter construct, and promoter activity was assayed in the RAW 264.7 macrophage cell line (Fig. 2). Previous experimental evidence has demonstrated that LPS is capable of inducing endogenous SOCS-1 expression in the RAW 264.7 cell line (5Nakagawa R. Naka T. Tsutsui H. Fujimoto M. Kimura A. Abe T. Seki E. Sato S. Takeuchi O. Takeda K. Akira S. Yamanishi K. Kawase I. Nakanishi K. Kishimoto T. Immunity. 2002; 17: 677-687Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Further, overexpression of SOCS-1 is also capable of inhibiting the LPS signaling pathway in the RAW 264.7 cell line (5Nakagawa R. Naka T. Tsutsui H. Fujimoto M. Kimura A. Abe T. Seki E. Sato S. Takeuchi O. Takeda K. Akira S. Yamanishi K. Kawase I. Nakanishi K. Kishimoto T. Immunity. 2002; 17: 677-687Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). LPS treatment elicited a moderate increase in the transcriptional activity starting with the –2523/–659 region of the SOCS-1 promoter (Fig. 2). The –945/–659 and –882/–659 regions of the SOCS-1 promoter demonstrated the most significant basal and LPS-induced activity. LPS stimulation yielded an approximate 7-fold increase in activity, which was diminished in promoter constructs lacking promoter sequence 3′ of the –864-position. The –768/–659 construct was transcriptionally unresponsive to LPS; nor did it demonstrate any significant basal promoter activity. The –768/–659 construct results were similar to the vector control lacking any SOCS-1 promoter sequence. Interestingly, we observed a significant decrease in promoter activity with the loss of promoter sequence between –850 and –840, indicating that this region of the promoter may contain an LPS-inducible cisacting element. These data indicate that the –864 to –659 region of SOCS-1 includes response elements required for the maximal LPS-induced transcriptional response of the promoter.Fig. 2Functional analysis of various regions of the SOCS-1 promoter. Shown to the left are the various regions cloned into the pGL3 basic luciferase plasmid. The number indicates the start of the region relative to the translational start site. All of the SOCS-1 promoter regions extend to +46 of the transcriptional start site (+1), which is denoted by the bent arrow. The vector control indicates the pGL3 basic plasmid
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