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siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
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Biologia
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JoVE Journal Biologia
siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

DOI:
10.3791/51572-v

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10:43 min

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May 24, 2014

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, , , ,
1MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences,University of Dundee, 2Centre for Gene Regulation and Expression, College of Life Sciences,University of Dundee

Capítulos

  • 00:05Título
  • 01:22Characterizing Hypoxia-responsiveness to Develop the Assay
  • 04:18Primary Screen: First Day
  • 07:27Primary Screen: Subsequent Days
  • 08:27Sample Screen
  • 10:09Conclusion

Summary

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Here, we describe a methodology to perform a targeted siRNA “ubiquitome” screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.  This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Tags

SiRNA ScreeningUbiquitinUbiquitin-like SystemBiological PathwaysMammalian CellsPost-translational ModificationUbiquitomeHIF1AHypoxia ResponseTranscription-based Luciferase ReporterAssay DevelopmentPrimary ScreeningSecondary ScreeningTertiary/deconvolution ScreeningTargeted SiRNA LibraryOff-target Effects

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