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Free keywords:
Endoplasmic Reticulum/metabolism
Flow Cytometry/*methods
Fluorescence
Genes, Reporter
Green Fluorescent Proteins/biosynthesis/genetics
HEK293 Cells
Humans
Proteasome Endopeptidase Complex/*genetics/*metabolism
Ubiquitin/chemistry/genetics/metabolism
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.
