Abstract
Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer's and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic membrane allowing a rapid influx of water leading to a loss of cellular integrity. As its role in human disease becomes apparent, methods identifying necroptosis will need to be further developed and optimized. Here we describe identification of necroptosis through quantifying cell death with pathway inhibitors and using western blots to identify end points of MLKL activation and protein-protein interactions leading to it.
Keywords:
Cell death; DSP-cross-linking; Dye exclusion; Immunoblots; Necroptosis; Vaccinia.
MeSH terms
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Acrylamides / pharmacology
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Animals
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Benzothiazoles / pharmacology
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Cell Line
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Cell Survival / drug effects
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Cross-Linking Reagents / chemistry
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Fibroblasts / drug effects
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Fibroblasts / metabolism
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Fibroblasts / virology*
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Fluorescent Dyes / chemistry
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Gene Expression Regulation
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Humans
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Imidazoles / pharmacology
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Immunoblotting / methods*
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Indoles / pharmacology
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Necroptosis / drug effects
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Necroptosis / genetics*
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Oligopeptides / pharmacology
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Phosphorylation / drug effects
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Protein Kinases / genetics*
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Protein Kinases / metabolism
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Protein Multimerization / drug effects
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Quinolines / pharmacology
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Receptor-Interacting Protein Serine-Threonine Kinases / antagonists & inhibitors
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Receptor-Interacting Protein Serine-Threonine Kinases / genetics*
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Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
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Succinimides / chemistry
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Sulfonamides / pharmacology
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Vaccinia virus / growth & development
Substances
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Acrylamides
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Benzothiazoles
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Cross-Linking Reagents
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Fluorescent Dyes
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GSK872
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Imidazoles
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Indoles
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N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide
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Oligopeptides
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Quinolines
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Succinimides
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Sulfonamides
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benzyloxycarbonyl-valyl-alanyl-aspartic acid
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necrostatin-1
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MLKL protein, mouse
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Protein Kinases
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Receptor-Interacting Protein Serine-Threonine Kinases
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Ripk1 protein, mouse
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Ripk3 protein, mouse
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dithiobis(succinimidylpropionate)