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A Flow Cytometric Assay for the Study of E3 Ubiquitin Ligase Activityb
dc.contributor.author | Hilliard, Joshua G. | |
dc.contributor.author | Cooper, Anne L. | |
dc.contributor.author | Slusser, Joyce G. | |
dc.contributor.author | Davido, David J. | |
dc.date.accessioned | 2017-06-09T16:07:04Z | |
dc.date.available | 2017-06-09T16:07:04Z | |
dc.date.issued | 2009-07 | |
dc.identifier.citation | Hilliard, J. G., Cooper, A. L., Slusser, J. G., & Davido, D. J. (2009). A Flow Cytometric Assay for the Study of E3 Ubiquitin Ligase Activity. Cytometry. Part A : The Journal of the International Society for Analytical Cytology, 75(7), 634–641. http://doi.org/10.1002/cyto.a.20738 | en_US |
dc.identifier.uri | http://hdl.handle.net/1808/24460 | |
dc.description | This is the peer reviewed version of the following article: Hilliard, J. G., Cooper, A. L., Slusser, J. G. and Davido, D. J. (2009), A flow cytometric assay for the study of E3 ubiquitin ligase activity. Cytometry, 75A: 634–641. doi:10.1002/cyto.a.20738, which has been published in final form at http://doi.org/10.1002/cyto.a.20738. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. | |
dc.description.abstract | BACKGROUND: Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. METHODS: A target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML-GFP fusion protein were selected by cell sorting and infected with an adenoviral vector expressing ICP0. RESULTS: In contrast to mock-infected cells, only PML-GFP-expressing cells infected with the ICP0 adenoviral vector led to a significant decrease in the fluorescence signal of PML-GFP when examined by fluorescence microscopy and FCM analysis. CONCLUSIONS: Using HSV-1 ICP0 as a paradigm, it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture with FCM analysis. | en_US |
dc.publisher | Wiley | en_US |
dc.subject | GFP | en_US |
dc.subject | Herpes simplex virus type 1 | en_US |
dc.subject | Infected cell protein 0 | en_US |
dc.subject | E3 ubiquitin ligase | en_US |
dc.subject | Promyelocytic leukemia | en_US |
dc.subject | FACS | en_US |
dc.title | A Flow Cytometric Assay for the Study of E3 Ubiquitin Ligase Activityb | en_US |
dc.type | Article | en_US |
kusw.kuauthor | Hilliard, Joshua G. | |
kusw.kuauthor | Cooper, Anne L. | |
kusw.kuauthor | Slusser, Joyce G. | |
kusw.kuauthor | Davido, David J. | |
kusw.kudepartment | Molecular Biosciences | en_US |
dc.identifier.doi | 10.1002/cyto.a.20738. | en_US |
kusw.oaversion | Scholarly/refereed, author accepted manuscript | en_US |
kusw.oapolicy | This item meets KU Open Access policy criteria. | en_US |
dc.identifier.pmid | PMC2750075 | en_US |
dc.rights.accessrights | openAccess |