Mihalas Anca B, Meffert Mollie K
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Methods Mol Biol. 2015;1280:61-74. doi: 10.1007/978-1-4939-2422-6_5.
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.
核因子κB（NF-κB）是一种强效转录因子，在中枢神经系统（CNS）中高度表达，在哺乳动物和无脊椎动物系统中，它已被证明是多种学习和记忆行为范式所必需的。NF-κB二聚体存在于神经元细胞体中，也存在于突触处，并可参与响应兴奋性神经传递的基因表达的活性依赖性调节。多个丝氨酸定向磷酸化事件在经典的NF-κB激活途径中至关重要，包括IκB激酶复合物（IKK）的激活以及NF-κB抑制剂（IκB）的磷酸化和降解。在本章中，我们描述了从解离培养的小鼠海马神经元中免疫沉淀（IP）IKK复合物的方法，随后进行体外激酶测定，通过监测GST-IκBα底物的磷酸化来评估兴奋性神经传递诱导的IKK激活。这些方法也可以在亚细胞减少的脑制备物中成功实施，例如生物化学分离的突触。
