U2OS cells were transfected with 500 ng of p73-luciferase or CDC6-luciferase plasmids, along with 500 ng of β-galactosidase and 2 μg of p3xFlag-CMV SENP7 V5 or empty vector for 48 hours. Cell extracts were then prepared in Reporter Lysis Buffer (Promega) and combined with luciferase reagent (Promega) for signal detection on a Microlumat Plus LB 96 V luminometer (Berthold Technologies). Alternatively, extract was mixed with β-galactosidase buffer [200 mM Na2PO4 (pH 7.3), 2 mM MgCl2, 100 mM β-mercaptoethanol, and ortho-nitrophenyl-galactosidase (1.33 mg/ml)] and incubated at 37°C before absorbance monitoring (415 nm) on a Sunrise microplate reader (Tecan). Reporter activity was determined from triplicate technical repeats as luciferase/β-galactosidase reading and expressed as fold induction compared to empty vector–expressing cells. Average (mean) fold changes with SE from two biological repeat experiments are shown.

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