The in vitro ubiquitination assay was carried out by using a ubiquitin-protein conjugation kit (Boston Biochem, Cambridge, MA). A rabbit anti-reticulocyte lysate-translated, 35S-labeled KLF5 antibody was used to observe the existence of GST-WWP1 or GST-Smurf2. The samples were subjected to 10% SDS-PAGE for autoradiography.
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