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Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases

  • Jan Hagenbucher
  • Hilda Stekman
  • Alfonso Rodriguez-Gil
  • Michael Kracht
  • M. Lienhard Schmitz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1510)

Abstract

The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.

Key words

Ubiquitin E3 ligases Proteasome Protein degradation Transfection Western blot qPCR 

Notes

Acknowledgements

The work from M.L.S. is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 1213) and Deutsche Krebshilfe (111447). The work of M.K. is supported from the Deutsche Forschungsgemeinschaft (Kr1143/5-3 and Kr1143/7-3). Both laboratories are further supported by SFB/TRR81, SFB1021, and the Excellence Cluster Cardio-Pulmonary System (ECCPS).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Jan Hagenbucher
    • 1
  • Hilda Stekman
    • 1
  • Alfonso Rodriguez-Gil
    • 1
  • Michael Kracht
    • 2
  • M. Lienhard Schmitz
    • 1
  1. 1.Medical Faculty, Institute of BiochemistryJustus-Liebig-UniversityGiessenGermany
  2. 2.Rudolf-Buchheim-Institute of PharmacologyJustus-Liebig-University GiessenGiessenGermany

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