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Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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Methods to Investigate EGFR Ubiquitination.

Alexia Conte1, Sara Sigismund2

  • 1IFOM, The FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139, Milan, Italy.

Methods in Molecular Biology (Clifton, N.J.)
|August 10, 2017
PubMed
Summary

Investigate epidermal growth factor receptor (EGFR) ubiquitination with biochemical methods. This chapter details in vitro assays, immunoprecipitation, ELISA, and immunofluorescence for studying EGFR signaling and degradation.

Keywords:
CblEGFRELISAEpidermal growth factor receptorImmunofluorescenceImmunoprecipitationIn vitro ubiquitination assayPhosphorylationUbiquitination

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Epidermal growth factor receptor (EGFR) ubiquitination is a key intracellular signal triggered by EGF stimulation.
  • This process regulates EGFR trafficking and ultimately targets the receptor for lysosomal degradation.

Purpose of the Study:

  • To provide an overview of biochemical methods for investigating EGFR ubiquitination.
  • To detail experimental protocols for assessing EGFR ubiquitination in vitro and in vivo.

Main Methods:

  • In vitro ubiquitination assay to replicate the EGFR ubiquitination milieu in a test tube.
  • Immunoprecipitation of EGFR followed by Western blot analysis to detect ubiquitinated forms.
  • ELISA-derived assay for robust assessment of EGFR ubiquitination from limited samples.
  • Immunofluorescence to visualize ubiquitinated EGFR within endocytic compartments post-EGF stimulation.

Main Results:

  • Established protocols for studying EGFR ubiquitination using various biochemical techniques.
  • Demonstrated methods for analyzing EGFR ubiquitination status and localization.
  • Highlighted the utility of different assays for varying sample amounts and research questions.

Conclusions:

  • Biochemical methods are crucial for understanding the role of EGFR ubiquitination in cellular signaling.
  • The described techniques enable comprehensive analysis of EGFR ubiquitination dynamics.
  • These methods facilitate research into EGFR-mediated cellular processes and disease mechanisms.