title: In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells.
abstract: This chapter deals with the fluorescence detection of SUMOylation and deSUMOylation in semi-intact cultured human cells, the so-called "in situ SUMOylation assay" and the "in situ deSUMOylation assay," respectively. In the in situ SUMOylation assay, the recombinant green-fluorescence protein fused to the SUMO1 (GFP-SUMO1) protein is used to visualize the nuclear rim, nucleolus, and nuclear bodies. These GFP signals represent cellular regions where SUMOylation efficiently takes place. If the recombinant SUMO-specific protease SENP1-catalytic domain is added after in situ SUMOylation, GFP signals can be erased. Therefore, the in situ SUMOylation assay can be used to assess deSUMOylation enzymatic activity.
authors.affiliation: Graduate School of Science and Technology, Kumamoto University, 2-3-1 Kurokami, Chu-o-ku, Kumamoto, 860-8555, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Yuasa
authors.affiliation: Graduate School of Science and Technology, Kumamoto University, 2-3-1 Kurokami, Chu-o-ku, Kumamoto, 860-8555, Japan. hisa@kumamoto-u.ac.jp.
authors.forename: Hisato
authors.initials: H
authors.lastname: Saitoh
mesh_terms: D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000201:enzymology* / Q000648:ultrastructure; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation
chemical_list: D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D049452:Green Fluorescent Proteins; D017830:Octoxynol; C404512:SENP1 protein, human; D003546:Cysteine Endopeptidases
keywords: Cell-based assay; Cell-permeabilization; DeSUMOylation; Fluorescence detection; SUMOylation