Methods to Investigate EGFR Ubiquitination.
Methods to Investigate EGFR Ubiquitination.
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DOI：
10.1007/978-1-4939-7219-7_5
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发表时间：
2017
影响因子：
--
通讯作者：
A. Conte;S. Sigismund
中科院分区：
文献类型：
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作者：
A. Conte;S. Sigismund
表皮生长因子受体（EGFR）的泛素化是一种重要的细胞内信号，在EGF刺激后发生，并在多个步骤控制EGFR的运输，最终决定受体的溶酶体降解。在本章中，我们给出了一个概述的生化方法来研究EGFR ubiquitination.First，我们描述了在体外ubiquitination测定，一种方法，在最小的ubiquitination机制的存在下，EGFR ubiquitination的生物环境是在试管中再现。在第二个协议中，我们解释了如何从总裂解物中免疫沉淀EGFR，并通过蛋白质印迹分析揭示其泛素化形式。然后，与ELISA衍生的测定，我们说明了一个强大的和可靠的方法来评估EGFR泛素化从低量的样品，最后，我们说明了一个免疫荧光协议，可视化泛素化的物种（包括EGFR本身）的EGFR阳性内吞隔室EGF刺激后。
Ubiquitination of the epidermal growth factor receptor (EGFR) is an important intracellular signal that occurs upon EGF stimulation and controls EGFR trafficking at multiple steps, finally destining the receptor to lysosomal degradation. In this chapter, we give an overview of the biochemical methods to investigate EGFR ubiquitination.Firstly, we describe the in vitro ubiquitination assay, a method where, in the presence of the minimal ubiquitination machinery, the biological milieu for EGFR ubiquitination is reproduced in a test tube. In the second protocol, we explain how to immunoprecipitate the EGFR from total lysate and reveal its ubiquitinated form by western blot analysis. Then, with an ELISA-derived assay, we illustrate a robust and reliable method to assess EGFR ubiquitination from low amount of sample; lastly, we illustrate an immunofluorescence protocol to visualize ubiquitinated species (including the EGFR itself) within the EGFR-positive endocytic compartments upon EGF stimulation.