title: Live-cell imaging of ubiquitin-proteasome system function.
abstract: The role of the ubiquitin-proteasome system (UPS) in maintaining protein homeostasis has generated a demand for assays that quantify UPS function in the presence of chemical and protein UPS inhibitors. Here, we describe protocols that measure changes in UPS reporter levels in response to changes in the expression level, localization, or aggregation state of a second protein. We utilize cell lines stably expressing fluorescent UPS substrates that are transfected with a second protein tagged with a compatible fluorophore. We describe protocols to correlate levels of UPS substrates with changes in the levels or properties of the transfected protein.
authors.affiliation: Department of Biology, Stanford University, Stanford, CA, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hipp
authors.forename: Kirill
authors.initials: K
authors.lastname: Bersuker
authors.forename: Ron R
authors.initials: RR
authors.lastname: Kopito
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease