Assaying Transcription Factor Stability

Jasmina Kurepa, Jan A. Smalle

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

10 Scopus citations

Abstract

Similar to the activities of transcription factors (TFs) in other eukaryotes, activities of many plant TFs are determined via regulated proteolysis by the ubiquitin/26S proteasome system. Thus, to fully understand the function of a TF, it is important to determine the fate of the active TF protein and unravel the environmental and intrinsic signals that control its total cellular level. Here we describe how to determine whether a TF of interest is targeted to the 26S proteasome for degradation. The given method combines analyses of the effects of translational inhibition and the inhibition of proteasome activity. An important requirement for these experiments is to monitor in parallel the effects of translational and proteasomal inhibition on the abundance of the TF and (1) on ubiquitin, which becomes rapidly depleted upon translational inhibition (2), on polyubiquitinated proteins, which accumulate upon successful inhibition of the 26S proteasome, and (3) on glutamine synthase, a very stable protein that is used as a general metabolic control. The method described here can be used to test TF stability under a variety of conditions and in different genetic backgrounds.

Original languageEnglish
Title of host publicationPlant Transcription Factors
Subtitle of host publicationMethods and Protocols
Pages219-234
Number of pages16
DOIs
StatePublished - 2011

Publication series

NameMethods in Molecular Biology
Volume754
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© 2011, Springer Science+Business Media, LLC.

Keywords

  • Immunoblot analyses
  • cycloheximide
  • glutamine synthase
  • proteasome inhibitor
  • transcription factor stability
  • ubiquitin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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