Advertisement

Notch Signaling Assays in Drosophila Cultured Cell Lines

  • Jinghua Li
  • Benjamin E. Housden
  • Sarah J. BrayEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)

Abstract

Signaling assays in Drosophila cell lines are a valuable method for investigating whether other proteins influence the function of the Notch pathway and for assessing whether specific enhancers or genes are regulated by Notch. In this chapter, we will describe two different types of assays that can be used to monitor Notch activation in Kc167 and S2 cells. One involves activating Notch in cultured cells and measuring the change in endogenous gene expression levels. The other uses luciferase reporters and measures their response to Notch, by co-transfecting with NICD.

Key words

Notch Drosophila cell line EDTA/EGTA treatment Real-time PCR E(spl) genes NRE Enhancer Dual-luciferase assay Luciferase reporter Cell transfection 

References

  1. 1.
    Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689PubMedCrossRefGoogle Scholar
  2. 2.
    Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Guruharsha KG, Kankel MW, Artavanis-Tsakonas S (2012) The Notch signalling system: recent insights into the complexity of a conserved pathway. Nat Rev Genet 13:654–666PubMedCrossRefGoogle Scholar
  4. 4.
    Rand MD, Grimm LM, Artavanis-Tsakonas S et al (2000) Calcium depletion dissociates and activates heterodimeric notch receptors. Mol Cell Biol 20:1825–1835PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Krejcí A, Bray S (2007) Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers. Genes Dev 21:1322–1327PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Goodfellow H, Krejcí A, Moshkin Y et al (2007) Gene-specific targeting of the histone chaperone asf1 to mediate silencing. Dev Cell 13:593–600PubMedCrossRefGoogle Scholar
  7. 7.
    Fiuza U-M, Klein T, Martinez Arias A et al (2010) Mechanisms of ligand-mediated inhibition in Notch signaling activity in Drosophila. Dev Dyn 239:798–805PubMedCrossRefGoogle Scholar
  8. 8.
    Bernard F, Krejcí A, Housden B et al (2010) Specificity of Notch pathway activation: twist controls the transcriptional output in adult muscle progenitors. Development 137:2633–2642PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Terriente-Felix A, Li J, Collins S et al (2013) Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme. Development 140:926–937PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Furriols M, Bray S (2001) A model Notch response element detects Suppressor of Hairless-dependent molecular switch. Curr Biol 11:60–64PubMedCrossRefGoogle Scholar
  11. 11.
    Bustin S, Bergkvist A, Nolan T (2011) In silico tools for qPCR assay design and data analysis. Methods Mol Biol 760:283–306PubMedCrossRefGoogle Scholar
  12. 12.
    Krejcí A, Bernard F, Housden BE et al (2009) Direct response to Notch activation: signaling crosstalk and incoherent logic. Sci Signal 2:ra1PubMedCrossRefGoogle Scholar
  13. 13.
    Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3–new capabilities and interfaces. Nucleic Acids Res 40:e115PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jinghua Li
    • 1
  • Benjamin E. Housden
    • 2
    • 1
  • Sarah J. Bray
    • 1
    Email author
  1. 1.Department of Physiology, Development and NeuroscienceUniversity of CambridgeCambridgeUK
  2. 2.Department of GeneticsHarvard Medical SchoolBostonUSA

Personalised recommendations