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Assay to Probe Proteolytic Processing of Notch by γ-Secretase

  • Lutgarde Serneels
  • Ina Tesseur
  • Bart De StrooperEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)

Abstract

With the increasing appreciation of the role of Notch in development and disease, measuring its cleavage and signaling activity in cellular systems has become important. Here we describe a cell-based method to analyze the cleavage of Notch at the S3 site by γ-secretase. HEK cells are transfected with an N-terminal truncated and myc-labeled mNotchΔE construct which can be easily and quantitatively detected by western blotting.

Key words

Notch signaling Notch cleavage NICD fragment Cell-based assay S3 cleavage γ-Secretase Immunoblot 

Notes

Acknowledgements

This work was supported by VIB and a Methusalem grant from KU Leuven and the Flemish Government. We would like to thank Tine Vanhoutvin for useful technical discussions and Veerle Vulsteke for drafting Fig. 1.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Lutgarde Serneels
    • 1
  • Ina Tesseur
    • 1
  • Bart De Strooper
    • 2
    Email author
  1. 1.VIB Center for the Biology of Disease - VIB11 and Center for Human GeneticsVIB and KU LeuvenLeuvenBelgium
  2. 2.VIB Center for the Biology of Disease and Center for Human Genetics and Institute of Neuroscience & Disease (LIND)KU Leuven and universitaire ziekenhuizenLeuvenBelgium

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