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A flow cytometric assay for the study of E3 ubiquitin ligase activity
Author(s) -
Hilliard Joshua G.,
Cooper Anne L.,
Slusser Joyce G.,
Davido David J.
Publication year - 2009
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20738
Subject(s) - ubiquitin ligase , ubiquitin , dna ligase , microbiology and biotechnology , biology , green fluorescent protein , fusion protein , ddb1 , flow cytometry , cell culture , protein degradation , biochemistry , dna , genetics , gene , recombinant dna
Abstract 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. For this study, 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, h erpes s implex v irus type 1 (HSV‐1) i nfected c ell p rotein 0 (ICP0) and one of its targets, p ro m yelocytic l eukemia (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. 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. Our results suggest that it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture by FCM analysis. © 2009 International Society for Advancement of Cytometry