TNF signaling: key protocols.
Tumor necrosis factor (TNF) is a pleiotropic cytokine that signals inflammation as well as cell death. We focus herein on the inflammatory pathway, giving particular emphasis to the in vitro methods used to study intracellular signaling mediators. The signal transduction cascade that TNF triggers after binding to the TNF receptors (TNFRs), flows throughout a series of protein-protein interactions as well as kinase activations, and finally leads to the translocation of distinct transcription factors within the nucleus, eventually inducing the transcription of specific inflammatory genes. In this chapter, we describe the analysis of the TNF receptor signaling complex by immunoprecipitation (IP), the activation of different MAP kinases by Western blot, the analysis of transcription factor activation by either electrophoretic mobility-shift assay (EMSA) or reporter assay, and the analysis of TNF-induced genes by chromatin IP (ChIP).
Muzio
Marta
M
DRO-Oncology, Pharmacology Department, Pharmacia, Nerviano, Italy.
Saccani
Simona
S
D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002843:Chromatin / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D015398:Signal Transduction; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D002843:Chromatin; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D001616:beta-Galactosidase