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Rab GTPases pp 305-317 | Cite as

Determining the Role of Rab7 in Constitutive and Ligand-Mediated Epidermal Growth Factor Receptor Endocytic Trafficking Using Single Cell Assays

  • Brian P. CeresaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)

Abstract

RAB proteins are essential for the proper membrane trafficking of a number of proteins. Each of the 60+ RABs that have been identified has a discrete role in coordinating the movement from one subcellular compartment to another. Early attempts at deciphering the roles of individual RAB proteins relied heavily on the use of activating and/or dominant negative mutants (Ceresa, Histol Histopathol 21:987–993, 2006). However, overexpression of mutant proteins can lead to misleading information; high levels of expression can drive low affinity (and possibly, nonphysiological) interactions as well as cause mislocalization. The use of RNAi for transient protein knock down will reveal which membrane trafficking steps absolutely require the attenuated RAB. When determining the role of RAB protein in epidermal growth factor receptor (EGFR) membrane trafficking, there are special considerations. The EGFR undergoes constitutive and ligand-mediated endocytic trafficking. Both affect receptor signaling, but via different mechanisms. Here, we discuss how to experimentally dissect those two processes.

Key words

siRNA EGFR Immunofluorescence Membrane trafficking RAB7 

Notes

Acknowledgments

This work was supported by NIH grant GM092874 and EY 021487. We thank members of the Ceresa Lab for critical reading of this manuscript, Jamie S. Rush for providing micrographs, and Sara Ceresa for photographic images.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Pharmacology and ToxicologyUniversity of LouisvilleLouisvilleUSA

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