Visualizing of signaling proteins on endosomes utilizing knockdown and reconstitution approach

Myoungkun Jeoung, Emilia Galperin

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

3 Scopus citations


Spatial distribution of intracellular signaling molecules and assembly of signaling complexes are yet to be fully understood. Studies of signaling events in time or space present a particular challenge due to the adverse effects that overexpression of signaling proteins may have on their functions and localization. To follow the distribution of signaling proteins in living cells we developed a methodology named knockdown and reconstitution (KDAR) that allows one to visualize proteins at levels of expression that are close to physiological. This methodology provides a stable expression of "endogenous" shRNA for long-term silencing of the targeted gene and simultaneous expression of a DNA cassette coding for a fluorescently labeled protein, which is insensitive to the targeting shRNA. In this chapter we discuss the needed reagents and outline two experimental approaches to generate KDAR stable cell lines. First, we demonstrate how the plasmid-mediated KDAR approach is successfully utilized to visualize spatial distribution of the GFP-labeled MEK2 in living cells. We then show how the lentivirus-mediated KDAR approach is used to reconstitute and visualize expression of the ERK1/2 scaffold protein Shoc2.

Original languageEnglish
Title of host publicationEndosome Signaling Part A
Number of pages17
StatePublished - 2014

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
This work was supported by grants from the National Cancer Institute (R00CA126161 to E. G.) and NIH Grant P20GM103486 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the NIGMS. We thank Dr. Mathew Gentry and Stacy Smith for critical reading of the manuscript and the Viral Production Core at the Department of Molecular and Cellular Biochemistry (University of Kentucky) for assistance with production of lentiviruses. The authors thank Maciej Wiznerowicz for valuable advice.


  • ERK1/2
  • Endocytosis
  • Fluorescence microscopy
  • MEK1/2
  • siRNA

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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