A Flow Cytometric Assay for the Study of E3 Ubiquitin Ligase Activityb
Hilliard, Joshua G.
Cooper, Anne L.
Slusser, Joyce G.
Davido, David J.
Scholarly/refereed, author accepted manuscript
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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.
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.
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
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