IKK kinase assay for assessment of canonical NF-κB activation in neurons.
Nuclear factor kappa B (NF-κB) is a potent transcription factor highly expressed in the central nervous system (CNS) where it has been shown to be required for multiple behavioral paradigms of learning and memory in both mammalian and invertebrate systems. NF-κB dimers are found in neuronal cell bodies, are also present at synapses, and can participate in the activity-dependent regulation of gene expression in response to excitatory neurotransmission. Multiple serine-directed phosphorylation events are critical in the canonical NF-κB activation pathway, including activation of the IκB kinase complex (IKK) and phosphorylation and degradation of the inhibitor of NF-κB (IκB). In this chapter, we describe methods for immunoprecipitation (IP) of the IKK complex from dissociated cultured murine hippocampal neurons, followed by in vitro kinase assay to evaluate excitatory neurotransmission-induced IKK activation by monitoring phosphorylation of a GST-IκBα substrate. These methods can also be successfully implemented in subcellular-reduced brain preparations, such as biochemically isolated synapses.
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Anca B
AB
Mihalas
Mollie K
MK
Meffert
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D057075:Enzyme Assays; D051550:I-kappa B Kinase / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D017966:Pyramidal Cells / Q000378:metabolism; D013379:Substrate Specificity
D016328:NF-kappa B; D051550:I-kappa B Kinase