In vitro Deubiquitination Assay

The DDB2-based ubiquitin E3 ligase (CRL-DDB2) was purified as described (Nishi et al., 2005; Sugasawa et al., 2005; Matsumoto et al., 2015). HA-tagged wild-type and catalytic mutant (C281A) USP44 was purified from two 10 cm dishes of MEFs transduced with the respective constructs using the HA tagged protein purification kit (MBL International Corporation, Woburn, MA, United States) according to the manufacturer’s recommendations (Zhang Y. et al., 2012). UbE1 and UBCH5a were obtained from Boston Biochem (Cambridge, MA, United States). CRL-DDB2 (40 ng) was ubiquitinated for 60 min at room temperature (21°C) by incubating UbE1 (200 ng), UBCH5a (800 ng), ubiquitin (10 μg) in a final volume of 30 μl of buffer containing 50 mM Tris-HCl pH 7.6, 10 mM MgCl2, 0.2 mM CaCl2, 4 mM ATP, 1 mM DTT, BSA (3 μg). When this reaction was completed, varying amounts of purified USP44 (2.5, or 5 μl from the 40 μl final volume of protein purified as above) and incubated for an additional 60 min at room temperature (21°C). This reaction was terminated by the addition of SDS sample buffer and boiling.