Methods to Study the Effect of IKK Inhibition on TNF-Inducing Apoptosis and Necroptosis in Cultured Cells.

The engagement of TNF on TNFR can result in cell survival or cell death depending on the different complex formation downstream this interaction. Here we describe reagents and assay procedures that can be used to study caspase-independent cell death (necroptosis) in cultured cells, in response to pharmacological interventions with NF-kappaB and death inhibitors. We provide protocol to detect death-specific proteins using immunoblot and to dissect necrosome complex by sequential co-immunoprecipitation of death-specific components during necroptosis.