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Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage

  • Sweta Iyer
  • Rachel T. Uren
  • Ruth M. Kluck
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization. Here we describe biochemical protocols that can be used on most cell types to gain a population overview of BAK and BAX status.

Key words

Apoptosis BAK BAX BCL-2 proteins Cytochrome c Disulfide linkage Mitochondria Oligomerization Pore formation 

Notes

Acknowledgment

This work was supported by NHMRC project grants 637337 and 1008434 and program grant 1016701, the Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC IRIISS.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sweta Iyer
    • 1
    • 2
  • Rachel T. Uren
    • 1
    • 2
  • Ruth M. Kluck
    • 1
    • 2
  1. 1.Molecular Genetics of Cancer DivisionThe Walter and Eliza Hall Institute of Medical ResearchParkvilleAustralia
  2. 2.Department of Medical BiologyThe University of MelbourneParkvilleAustralia

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