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A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3
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JoVE Journal Biology
A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

DOI:
10.3791/53723-v

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11:44 min

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January 24, 2016

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, , ,
1CRCHUM – Research center, Centre Hospitalier de l’Université de Montréal,Université de Montréal, 2Department of Biochemistry and Molecular Medicine,Université de Montréal, 3Faculty of Medicine,Université de Montréal

Chapters

  • 00:05Title
  • 01:01Infection of A549 Cells
  • 03:36Preparation of Whole Cell Extracts
  • 05:07Resolution of Whole Cell Extracts by High Resolution SDS-PAGE
  • 06:20Analysis of IRF3 Dimerization by Native-PAGE
  • 07:37Immunoblot Analysis of IRF3 Species
  • 09:23Results: Detection Distinct Phosphorylated IRF3 Species
  • 10:56Conclusion

Summary

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Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

Tags

High Resolution MethodMonitorPhosphorylation-dependent ActivationIRF3Transcription FactorInnate Immune ResponseInterferon Beta ExpressionPathogensPathogen-associated Molecular PatternsA549 CellsCultureInfectionTrypsinizeCentrifugeCell PelletSingle-cell Suspension

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