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).
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Acknowledgments
The authors would like to thank Dr. Andreas Gewies for critical reading of the manuscript and all members of the laboratory for input and discussion.
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Gorka, O., Wanninger, S., Ruland, J. (2014). Detection of NF-κB Pathway Activation in T Helper Cells. In: Waisman, A., Becher, B. (eds) T-Helper Cells. Methods in Molecular Biology, vol 1193. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1212-4_8
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DOI: https://doi.org/10.1007/978-1-4939-1212-4_8
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1211-7
Online ISBN: 978-1-4939-1212-4
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