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

Abstract

(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.