title: Detection of NF-κB pathway activation in T helper cells.
abstract: Upon activation of CD4+ T helper cells the transcription factor complex NF-κB becomes activated and its subunits are able to translocate to the nucleus. There, the dimerized proteins regulate the transcription of for example cytokine and pro-survival genes. This process is of central importance for a proper function of T helper cells as its loss causes severe immunodeficiency, whereas its deregulation can contribute to lymphomagenesis or autoimmunity. In this protocol we describe four methods to investigate NF-κB activation in T helper cells by testing (1) the assembly of the Carma1-Malt1-Bcl10 complex by co-immunoprecipitation, (2) the activation of the IKK complex and the degradation of IκBα by western blot analysis, (3) the degradation of IκBα by intracellular flow cytometry, and (4) the nuclear translocation and DNA binding activity of NF-κB by nonradioactive electrophoretic mobility shift assay (EMSA).
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, 81675, Munich, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Gorka
authors.forename: Stefan
authors.initials: S
authors.lastname: Wanninger
authors.forename: Jürgen
authors.initials: J
authors.lastname: Ruland
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D000074018:B-Cell CLL-Lymphoma 10 Protein; D053468:CARD Signaling Adaptor Proteins / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation / Q000502:physiology; D051379:Mice; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; D016328:NF-kappa B / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D055503:Protein Multimerization; D015398:Signal Transduction / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000166:cytology / Q000276:immunology / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000074018:B-Cell CLL-Lymphoma 10 Protein; C118591:BCL10 protein, human; D053468:CARD Signaling Adaptor Proteins; D016328:NF-kappa B; D009363:Neoplasm Proteins; D020169:Caspases; C400109:MALT1 protein, human; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; C426242:CARD11 protein, human; D006162:Guanylate Cyclase