Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells

Pavel Davidovich; Seamus J Martin

doi:10.1016/j.xpro.2024.103126

Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells

STAR Protoc. 2024 Sep 20;5(3):103126. doi: 10.1016/j.xpro.2024.103126. Epub 2024 Jul 31.

Authors

Pavel Davidovich¹, Seamus J Martin²

Affiliations

¹ Molecular Cell Biology Laboratory, The Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland.
² Molecular Cell Biology Laboratory, The Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland. Electronic address: [email protected].

Abstract

Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.¹.

Keywords: cell biology; immunology; protein biochemistry.

MeSH terms

Caspase 8 / metabolism
Cell Line
Fas Ligand Protein / metabolism
Fas-Associated Death Domain Protein* / metabolism
Humans
Immunoprecipitation* / methods
NF-kappa B / metabolism
Signal Transduction*
TNF-Related Apoptosis-Inducing Ligand* / metabolism
TNF-Related Apoptosis-Inducing Ligand* / pharmacology
fas Receptor* / metabolism

Substances

TNF-Related Apoptosis-Inducing Ligand
Fas-Associated Death Domain Protein
fas Receptor
Fas Ligand Protein
TNFSF10 protein, human
Caspase 8
FADD protein, human
FAS protein, human
NF-kappa B