Detection of RIPK1 in the FADD-Containing Death Inducing Signaling Complex (DISC) During Necroptosis.
Detection of RIPK1 in the FADD-Containing Death Inducing Signaling Complex (DISC) During Necroptosis.
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DOI：
10.1007/978-1-4939-8754-2_10
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发表时间：
2018
影响因子：
--
通讯作者：
Rosalind L. Ang;A. Ting
中科院分区：
文献类型：
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作者：
Rosalind L. Ang;A. Ting
具有死亡结构域的Fas相关蛋白(FADD)是肿瘤坏死因子受体超家族(TNFRSF)成员如Fas和TNFR1等诱导细胞凋亡所必需的信号分子。FADD是一种小的适配分子，其功能是充当招募Proaspase-8和其他调节剂的支架。启动细胞凋亡级联反应的含有FADD的信号复合体被称为死亡诱导信号复合体(DISC)。在没有FADD的情况下，死亡受体不能诱导细胞凋亡，在适当的细胞类型中，这些死亡受体继而诱导坏死性下垂。当caspase-8被抑制时，FADD充足的细胞中的死亡受体也可以诱导坏死性下垂，通常是通过添加caspase抑制剂来实现的。在这种坏死性条件下，FADD的免疫沉淀分离圆盘可以用来检测这种复合体的成分。在这里，我们描述了FADD的免疫沉淀和随后的Western-blotting来确定在坏死性下垂期间这个复合体中的RIPK1。
FAS-associated protein with death domain (FADD) is a signaling molecule required by members of the TNF receptor superfamily (TNFRSF) such as FAS and TNFR1 to induce apoptosis. FADD is a small adapter molecule that functions as a scaffold to recruit procaspase-8 and other regulators. The FADD-containing signaling complex that initiates the apoptotic cascade has been termed the death inducing signaling complex (DISC). In the absence of FADD, death receptors cannot induce apoptosis and in appropriate cell types, these death receptors then induce necroptosis. Necroptosis can also be induced by death receptors in FADD-sufficient cells when caspase-8 is inhibited, usually accomplished by the addition of caspase inhibitors. Under such necroptotic conditions, the immunoprecipitation of FADD to isolate the DISC can be utilized to examine components of this complex. Here, we describe the immunoprecipitation of FADD and subsequent western-blotting to identify RIPK1 in this complex during necroptosis.