Abstract
The Suppressor of Cytokine Signalling (SOCS) proteins are a family of negative regulators characterized by a central SH2 domain and C-terminal SOCS box motif. Cytokine Inducible SH2-containing protein (CIS), SOCS1, 2 and 3 are rapidly upregulated in response to cytokine stimulation and act to inhibit JAK/STAT signalling by a variety of mechanisms. The expression of SOCS proteins provides a level of specificity in the control of signalling, with SOCS proteins differentially upregulated in response to individual cytokines and in various cell-types. Real-time reverse transcription (RT) quantitative polymerase chain reaction (RT-qPCR) is an established technique for quantifying mRNA in biological samples, measuring the relative expression of genes of interest and identifying single nucleotide polymorphisms. Here we describe the use of SYBR® Green I RT-qPCR to quantify the relative expression level of SOCS mRNA in murine bone marrow-derived macrophages (BMDM). The approach can be universally applied to different cell types and various tissues.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Starr R, Willson TA, Viney EM et al (1997) A family of cytokine-inducible inhibitors of signalling. Nature 387:917–921
Hilton DJ, Richardson RT, Alexander WS 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
Wormald S, Zhang JG, Krebs DL et al (2006) The comparative roles of suppressor of cytokine signaling-1 and -3 in the inhibition and desensitization of cytokine signaling. J Biol Chem 281:11135–11143
Matsumoto A, Masuhara M, Mitsui K et al (1997) CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation. Blood 89:3148–3154
Greenhalgh CJ, Rico-Bautista E, Lorentzon M et al (2005) SOCS2 negatively regulates growth hormone action in vitro and in vivo. J Clin Invest 115:397–406
Yasukawa H, Misawa H, Sakamoto H et al (1999) The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J 18: 1309–1320
Sasaki A, Yasukawa H, Suzuki A et al (1999) Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells 4:339–351
Babon JJ, Kershaw NJ, Murphy JM, et al (2012) SOCS3 binds to a site unique to JAKs and inhibits their kinase activity via a novel, non-competitive mechanism. Immunity. In Press
Zhang JG, Farley A, Nicholson SE et al (1999) The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc Natl Acad Sci U S A 96:2071–2076
Kamura T, Sato S, Haque D et al (1998) The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev 12:3872–3881
Babon JJ, Sabo JK, Zhang JG et al (2009) The SOCS box encodes a hierarchy of affinities for Cullin5: implications for ubiquitin ligase formation and cytokine signalling suppression. J Mol Biol 387:162–174
Yasukawa H, Ohishi M, Mori H et al (2003) IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat Immunol 4:551–556
Dickensheets H, Vazquez N, Sheikh F et al (2007) Suppressor of cytokine signaling-1 is an IL-4-inducible gene in macrophages and feedback inhibits IL-4 signaling. Genes Immun 8:21–27
Yoshikawa H, Matsubara K, Qian GS et al (2001) SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 28:29–35
Tamiya T, Kashiwagi I, Takahashi R et al (2011) Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: regulation of T-cell inflammation by SOCS1 and SOCS3. Arterioscler Thromb Vasc Biol 31:980–985
Akhtar LN, Benveniste EN (2011) Viral exploitation of host SOCS protein functions. J Virol 85:1912–1921
Mullis K, Faloona F, Scharf S et al (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51(Pt 1):263–273
Saiki RK, Scharf S, Faloona F et al (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354
Higuchi R, Fockler C, Dollinger G et al (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 11:1026–1030
Huggett J, Dheda K, Bustin S et al (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284
Radonic A, Thulke S, Mackay IM et al (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862
Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034
Cook NL, Vink R, Donkin JJ et al (2009) Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury. J Neurosci Res 87:34–41
Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30(6):503–512.
Acknowledgments
This research was supported by the NHMRC Australia (Program Grant 461219 and fellowship to S.E.N.) and an NIH Grant (CA022556). It was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kolesnik, T.B., Nicholson, S.E. (2013). Analysis of Suppressor of Cytokine Signalling (SOCS) Gene Expression by Real-Time Quantitative PCR. In: Nicholson, S., Nicola, N. (eds) JAK-STAT Signalling. Methods in Molecular Biology, vol 967. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-242-1_17
Download citation
DOI: https://doi.org/10.1007/978-1-62703-242-1_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-241-4
Online ISBN: 978-1-62703-242-1
eBook Packages: Springer Protocols