Methods to Investigate EGFR Ubiquitination

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1652)

Abstract

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.

Key words

Epidermal growth factor receptor EGFR Ubiquitination Cbl Phosphorylation In vitro ubiquitination assay Immunoprecipitation ELISA Immunofluorescence 
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Notes

Acknowledgments

The authors gratefully acknowledge Rosalind Gunby for critically reviewing this manuscript and Roberta Pascolutti for the EGFR in vitro ubiquitination experiment. SS was supported by a grant from Worldwide Cancer Research (16-1245). AC was supported by FIRC Fellowship 14909.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.IFOM, The FIRC Institute for Molecular Oncology FoundationMilanItaly

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