Chapter title |
Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases
|
---|---|
Chapter number | 22 |
Book title |
HDAC/HAT Function Assessment and Inhibitor Development
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6527-4_22 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6525-0, 978-1-4939-6527-4
|
Authors |
Jan Hagenbucher, Hilda Stekman, Alfonso Rodriguez-Gil, Michael Kracht, M. Lienhard Schmitz |
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. |
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