A flow cytometric assay for the study of E3 ubiquitin ligase activity.
A flow cytometric assay for the study of E3 ubiquitin ligase activity.
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DOI：
10.1002/cyto.a.20738
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发表时间：
2009-07
期刊：
影响因子：
3.7
通讯作者：
Davido, David J.
中科院分区：
文献类型：
--
作者：
Hilliard, Joshua G.;Cooper, Anne L.;Slusser, Joyce G.;Davido, David J.
关键词：
目前用于监测细胞培养物或体内E3泛素连接酶活性的方法是有限的。因此，许多E3泛素连接酶在活细胞中对细胞靶点的降解尚未被研究。E3泛素连接酶的靶标在细胞培养物中表达为荧光标记的蛋白质。如果E3泛素连接酶介导细胞培养物中靶蛋白的降解，则通过FCM分析预期靶将显示降低的荧光信号。我们最初使用E3泛素连接酶，单纯疱疹病毒1型（HSV-1）感染的细胞蛋白0（ICP 0）和它的目标之一，早幼粒细胞白血病（PML）蛋白，以确定我们的方法的可行性。通过细胞分选选择表达PML-GFP融合蛋白的细胞，并用表达ICP 0的腺病毒载体感染。与模拟感染的细胞相比，只有PML-GFP表达细胞感染的ICP 0腺病毒载体导致PML-GFP的荧光信号显着减少时，通过荧光显微镜和FCM分析检查。使用HSV-1 ICP 0作为范例，可以用FCM分析检查细胞培养物中E3泛素连接酶（通过其靶标之一）的活性。
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. 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. 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. 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.
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DOI：
10.1152/ajprenal.90482.2008
发表时间：
2008-11-01
影响因子：
4.2
作者：
Chen, Guangping;Huang, Haidong;Sands, Jeff M.
通讯作者：
Sands, Jeff M.
影响因子：
11.4
作者：
EVERETT, RD
通讯作者：
EVERETT, RD
影响因子：
5.4
作者：
Everett, RD;Zafiropoulos, A
通讯作者：
Zafiropoulos, A
影响因子：
3.8
作者：
MAUL, GG;GULDNER, HH;SPIVACK, JG
通讯作者：
SPIVACK, JG
DOI：
10.1073/pnas.242594299
发表时间：
2002-11-26
影响因子：
11.1
作者：
Paulmurugan, R;Umezawa, Y;Gambhir, SS
通讯作者：
Gambhir, SS