Investigating Ubiquitination and Deubiquitination of MDM2 and NFATc2

Total cell lysates or subcellular extracts were prepared and subjected to IB assays as described^{41 (link)}. The Santa Cruz anti-USP15 (2D5) and the Proteintech anti-USP15 antibodies were used for detecting human and mouse USP15, respectively. The Millipore anti-MDM2 and the Sigma anti-MDM2 antibodies were used for detecting human and mouse MDM2, respectively. For ubiquitination assays, MDM2 and NF-ATc2 were isolated by IP under denaturing conditions^{39 (link)}, and the ubiquitinated MDM2 and NFATc2 were detected by IB using a ubiquitin antibody specifically detecting K48-linked ubiquitin chains (Millipore). For transfection models, MDM2 or NFATc2 was transfected into HEK293 cells along with HA-ubiquitin, and the ubiquitinated MDM2 and NFATc2 were isolated by denaturing IP and detected by anti-HA IB. For deubiquitination assays, the isolated MDM2–ubiquitin conjugates were incubated with purified USP15 or its catalytic mutant in a deubiquitination buffer^{42 (link)} for 4 h and then subjected to IB using anti-ubiquitin.