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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
 

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Article DOI: 10.3791/3327-v • 10:25 min • November 10th, 2012
November 10th, 2012 •
1, 1, 1, 2, 1
1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center

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A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

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Ubiquitin-proteasome Activity Degron Green Fluorescent Protein (GFP) Reporter Protein Proteolysis Intracellular Organelle Abnormal Proteins Misfolded Proteins Damaged Proteins Oxidized Proteins Proteasome Maintenance Cellular Processes Stress Response Cell Cycle Regulation Cellular Differentiation Immune System Response Ubiquitin-proteasome System Dysfunction Tumors Neurodegenerative Diseases Cellular Senescence Organismal Aging Primary Cells Lentiviral Expression Vectors Transfection Efficiency
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