A method for the quantitative analysis of stimulation-induced nuclear translocation of the p65 subunit of NF-κB from patient-derived dermal fibroblasts.
A method for the quantitative analysis of stimulation-induced nuclear translocation of the p65 subunit of NF-κB from patient-derived dermal fibroblasts.
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DOI：
10.1007/978-1-4939-2422-6_25
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Hanson EP
中科院分区：
文献类型：
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作者：
Wessel AW;Hanson EP
发育性和免疫介导的疾病与参与NF-κ B活化的关键信号传导组分的基因突变有关，导致经典IKK复合物的活化或调节受损。我们通过临床表型分析和基因测序鉴定疑似或已知NF-κ B信号通路缺陷的患者。为了帮助理解突变是如何导致疾病的，我们定量了细胞中NF-κ B激活信号事件的动力学和剂量反应。经典IKK复合物激活后，NF-κ B蛋白抑制剂（I κ B）的磷酸化导致其降解，随后NF-κ B家族成员从细胞质易位至细胞核。在这里，我们提供了一种方法，以获得患者来源的皮肤成纤维细胞和定量评估的完整性的信号转导途径，从受体活化核p65易位。
Developmental and immune-mediated disease has been linked to genetic mutation of key signaling components involved in NF-κB activation that leads to impaired activation or regulation of the canonical IKK complex. We identify patients with suspected or known defects of the NF-κB signaling pathway through clinical phenotyping and genetic sequencing. To help understand how mutations cause disease, we quantitate the kinetics and dose-response of NF-κB activation signaling events in their cells. Following activation of the canonical IKK complex, phosphorylation of the inhibitor of NF-κB proteins (IκB) leads to their degradation and the subsequent translocation of NF-κB family members from the cell cytoplasm to the nucleus. Here, we provide a method to obtain patient-derived dermal fibroblasts and quantitatively assess the integrity of the signal transduction pathway from receptor activation to nuclear p65 translocation.