title: Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases.
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.
authors.affiliation: Medical Faculty, Institute of Biochemistry, Justus-Liebig-University, Friedrichstrasse 24, Giessen, 35392, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Hagenbucher
authors.affiliation: Medical Faculty, Institute of Biochemistry, Justus-Liebig-University, Friedrichstrasse 24, Giessen, 35392, Germany.
authors.forename: Hilda
authors.initials: H
authors.lastname: Stekman
authors.affiliation: Medical Faculty, Institute of Biochemistry, Justus-Liebig-University, Friedrichstrasse 24, Giessen, 35392, Germany.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Rodriguez-Gil
authors.affiliation: Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University Giessen, Giessen, D-35392, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kracht
authors.affiliation: Medical Faculty, Institute of Biochemistry, Justus-Liebig-University, Friedrichstrasse 24, Giessen, 35392, Germany. lienhard.schmitz@biochemie.med.uni-giessen.de.
authors.forename: M Lienhard
authors.initials: ML
authors.lastname: Schmitz
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D050880:p300-CBP Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000098563:Seven in Absentia Proteins; D000099224:p300-CBP-Associated Factor
chemical_list: D009687:Nuclear Proteins; D046988:Proteasome Endopeptidase Complex; D034741:RNA, Small Interfering; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D050880:p300-CBP Transcription Factors; D000098563:Seven in Absentia Proteins; D000099224:p300-CBP-Associated Factor
keywords: Proteasome; Protein degradation; Transfection; Ubiquitin E3 ligases; Western blot; qPCR