Notch signaling is a highly conserved signaling pathway, which is critical for many cell fate decisions. Ligand activation of Notch leads to cleavage of the Notch receptor and liberation of the Notch intracellular domain (ICD) from the membrane-tethered receptor. After translocation to the nucleus, the Notch ICD interacts with the DNA-binding protein CSL to activate gene transcription. To better understand the temporal and spatial aspects of Notch signaling, we here describe a fluorescent protein-based reporter assay that allows Notch activation to be followed in real time in individual cells. We have generated a reporter construct composed of 12 CSL-binding motifs linked to fluorescent proteins with different half-lives: a stabler red fluorescent protein (DsRedExpressDR) and a destabilized form of green fluorescent protein (d1EGFP). The fluorescent reporters reflect the activation status of Notch signaling with single-cell resolution. The reporters rapidly respond to various forms of Notch activation, including ligand activation of full-length Notch receptors. Finally, we use this assay to gain insights into the level of Notch signaling in CNS progenitor cells in culture and in vivo.

This content is only available via PDF.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.