title: Ripoptosome Analysis by Caspase-8 Coimmunoprecipitation.
abstract: The biochemical signaling of cell death pathways is executed at a number of different intracellular and/or membrane-bound high-molecular mass complexes. It is crucial to be able to detect the formation, differences in assembly, and differential composition of such complexes to understand their contribution to the execution phase of apoptotic or necroptotic cell death. We describe here the use of caspase-8 coimmunoprecipitation in the spontaneously transformed keratinocyte cell line, HaCaT, to study the formation and composition of the Ripoptosome, a complex that is based on the serine-threonine kinase receptor-interacting protein 1 (RIPK1). However, the method can be adapted for use with other antibodies and cell lines. This protocol determines whether cells form the Ripoptosome complex, which is important for both apoptosis and necroptosis execution. Caspase-8 is an indispensible Ripoptosome component; therefore, caspase-8 antibodies are used to pull down the respective complex. However, the method cannot discriminate whether this complex triggers apoptosis (through the RIPK1 → FADD → caspase-8 activation pathway), necroptosis (through the RIPK1 → RIPK3 → MLKL activation pathway) or nondeath signaling. The actual signaling output (death or nondeath signaling) depends on the stoichiometry of the respective molecules as well as on the activity of FLIP, caspase-8, or other factors.
authors.affiliation: Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University Heidelberg, Heidelberg 68167, Germany;
authors.forename: Maria
authors.initials: M
authors.lastname: Feoktistova
authors.affiliation: Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University Heidelberg, Heidelberg 68167, Germany;
authors.forename: Peter
authors.initials: P
authors.lastname: Geserick
authors.affiliation: Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University Heidelberg, Heidelberg 68167, Germany; Department of Dermatology & Allergology, University Hospital of RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Leverkus
mesh_terms: D053181:Caspase 8 / Q000032:analysis* / Q000276:immunology; D016923:Cell Death; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D015603:Keratinocytes / Q000737:chemistry / Q000502:physiology; D046911:Macromolecular Substances / Q000302:isolation & purification*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000032:analysis*
chemical_list: D046911:Macromolecular Substances; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D053181:Caspase 8