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Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses   

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Original research article

A brief version of this protocol appeared in:
PLOS Pathogens
Dec 2012

Abstract

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin immunoprecipitation (ChIP) assays, electrophoresis mobility shift assays (EMSA), streptavidin-agarose mediated DNA pull-down assays, together with cell-based transcription reporter assays were conducted to verify whether the query protein is involved in EBNA2-dependent transcription. The ATP-bound state of nuclear chaperone nucleophosmin (NPM1) has been implicated in pleiotropic biological processes. An ATP-agarose-mediated pull-down protocol was developed to monitor the formation of the pre-initiation complex that is induced by ATP-bound NPM1. According to EBNA2 and Notch-IC have been shown to be partially interchangeable with respect to activation of target genes in B cell lines, it is conceivable that EBNA2 is a biological equivalent of an activated Notch IC.

Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liu, C., Chen, Y., Min, Y., Zhao, B., Cheng, C., Kang, M., Chiu, S., Kieff, E. and Peng, C. (2013). Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses. Bio-protocol 3(18): e901. DOI: 10.21769/BioProtoc.901.
  2. Liu, C. D., Chen, Y. L., Min, Y. L., Zhao, B., Cheng, C. P., Kang, M. S., Chiu, S. J., Kieff, E. and Peng, C. W. (2012). The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells. PLoS Pathog 8(12): e1003084.
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