Conte Alexia, Sigismund Sara
IFOM, The FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139, Milan, Italy.
Methods Mol Biol. 2017;1652:81-100. doi: 10.1007/978-1-4939-7219-7_5.
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.
表皮生长因子受体（EGFR）的泛素化是一种重要的细胞内信号，在表皮生长因子（EGF）刺激时发生，并在多个步骤中控制EGFR的运输，最终使该受体走向溶酶体降解。在本章中，我们概述了研究EGFR泛素化的生化方法。首先，我们描述体外泛素化测定法，即在存在最小泛素化机制的情况下，在试管中重现EGFR泛素化的生物学环境的方法。在第二个方案中，我们解释如何从总裂解物中免疫沉淀EGFR，并通过蛋白质免疫印迹分析揭示其泛素化形式。然后，通过一种源自酶联免疫吸附测定（ELISA）的方法，我们阐述一种从少量样品中评估EGFR泛素化的稳健且可靠的方法；最后，我们阐述一种免疫荧光方案，以在EGF刺激后可视化EGFR阳性内吞小室中的泛素化物质（包括EGFR本身）。
