title: PPAR SUMOylation: some useful experimental tips.
abstract: Studies on the regulation of nuclear receptors, such as the peroxisome proliferator-activated receptors (PPARs), are important to enhance our understanding of their molecular, cellular, and physiological behavior. A decade ago, it was shown that the SUMOylation pathway plays a very important role in the regulation of transcription factor activity. The SUMOylation process involves the covalent binding of SUMO protein to the target protein. However, experimental procedures to demonstrate that low-expressed proteins, such as PPARs, are SUMOylated, remain tricky, and require specific optimization for each protein.Here, we provide a simple and useful experimental method to investigate the SUMOylation of PPARs in a cellular context. The procedure for studying SUMOylation in living cells is based on the purification under denaturating conditions of total SUMOylated proteins followed by the specific detection of the PPAR proteins. For that purpose, cells are transfected with both 6xHistidine-tagged SUMO and PPAR expression vectors. Since the polyHistidine tag binds to nickel cationic ion-linked agarose matrix (Ni-NTA matrix), His-tagged SUMO proteins covalently linked to the protein substrate can be specifically precipitated and separated from the unSUMOylated proteins. The SUMO-modified PPAR proteins can subsequently be visualized by western blotting using anti-PPAR antibodies. Many questions relative to the regulation of PPAR SUMOylation can be appropriately addressed by adapting this protocol.
authors.affiliation: Center for Clinical Pharmacology, Division of Medicine, University College of London, London, UK.
authors.forename: Benoit
authors.initials: B
authors.lastname: Pourcet
authors.forename: Bart
authors.initials: B
authors.lastname: Staels
authors.forename: Corine
authors.initials: C
authors.lastname: Glineur
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D019295:Computational Biology; D006801:Humans; D047492:Peroxisome Proliferator-Activated Receptors / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D014162:Transfection
chemical_list: D047492:Peroxisome Proliferator-Activated Receptors