IKK kinase assay for assessment of canonical NF-κB activation in neurons.
IKK kinase assay for assessment of canonical NF-κB activation in neurons.
复制标题
IKK激酶分析用于评估神经元中的典型NF-κB激活。
DOI：
10.1007/978-1-4939-2422-6_5
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Meffert MK
中科院分区：
文献类型：
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作者：
Mihalas AB;Meffert MK
核因子κ B（NF-κB）是一种在中枢神经系统（CNS）中高度表达的转录因子，在哺乳动物和无脊椎动物的学习和记忆的多种行为模式中都需要它。NF-κB二聚体存在于神经元细胞体中，也存在于突触中，并且可以参与响应兴奋性神经传递的基因表达的活性依赖性调节。多个丝氨酸指导的磷酸化事件在经典NF-κB活化途径中是关键的，包括IκB激酶复合物（IKK）的活化以及NF-κB抑制剂（IκB）的磷酸化和降解。在本章中，我们描述了分离培养的小鼠海马神经元的IKK复合物的免疫沉淀（IP）方法，然后通过监测GST-I κBα底物的磷酸化，体外激酶测定来评估兴奋性神经传递诱导的IKK激活。这些方法也可以成功地在亚细胞减少的脑制备中实施，例如生化分离的突触。
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.