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（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测
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JoVE Journal Biology
Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
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（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

DOI:
10.3791/3327-v

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10:25 min

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November 10, 2012

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, , , ,
1Molecular and Cell Biology,Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging,Leiden University Medical Center

Chapters

  • 00:00Title
  • 01:21Virus Production
  • 04:11Titering of Virus
  • 06:13Transduction of Human Diploid Fibroblasts
  • 07:19FACS Experiments
  • 08:50Proteasomal Activity in vivo
  • 10:04Conclusion

Summary

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Automatic Translation

监控活细胞的泛素 - 蛋白酶体活性的方法。一个degron不稳定的绿色荧光蛋白（GFP-DGN）和一个稳定的的GFP-dgnFS融合蛋白的生成和使用慢病毒表达载体导入细胞。这种技术允许产生稳定的GFP-dgn/GFP-dgnFS表达细胞株中，泛素 - 蛋白酶体活性可以很容易地使用的反射荧光或流式细胞术评估。

Tags

Ubiquitin-proteasome ActivityDegronGreen Fluorescent Protein (GFP)Reporter ProteinProteolysisIntracellular OrganelleAbnormal ProteinsMisfolded ProteinsDamaged ProteinsOxidized ProteinsProteasome MaintenanceCellular ProcessesStress ResponseCell Cycle RegulationCellular DifferentiationImmune System ResponseUbiquitin-proteasome System DysfunctionTumorsNeurodegenerative DiseasesCellular SenescenceOrganismal AgingPrimary CellsLentiviral Expression VectorsTransfection Efficiency

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