Poinb amsh

A plasmid for expression of the AMSH* deubiquitylating enzyme (pOINB-AMSH*)^{38 (link)} was obtained from AddGene (Plasmid #66712). AMSH* expression was induced in 1 L Rosetta 2 (DE3) pLysS cells (Novagen) with 0.4 mM IPTG for 18 hr at 18°C. Bacterial cell pellets were suspended in binding buffer (20 mM Tris-HCl [pH 8.5], 300 mM NaCl, 50 mM imidazole, 2 mM β-mercaptoethanol, and 1× Roche cOmplete protease inhibitor cocktail) and sonicated. The soluble lysate was collected following centrifugation at 25,000 rpm in a SW40.1 rotor for 1 hr and bound to TALON resin (Clonetech) for 90 min at 4°C. The bound resin was then washed with 1 L binding buffer and AMSH* was eluted with binding buffer containing 250 mM imidazole. The eluate was then concentrated and dialyzed against 20 mM Trsi-HCl (pH 8.5), 150 mM NaCl, 4 mM DTT at 4°C and 6 µM aliquots were stored at −80°C. Analysis of ubiquitin chains on MCM7 and MCM4 was performed using UbiCrest deubiquitylating enzymes (Boston Biochem). Plasmid pull downs were performed as described above, except that the beads were resuspended in 1× DUB reaction buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 5 mM DTT) and then incubated with 5 µM USP2_CD, 8 µM Otubain1 (OTUB1), 0.6 µM AMSH*, 1× Yod1, 1× OTUD3, 1× Trabid, 1× Cezanne, or 1× Otulin for 30 min at 37°C. Reactions were quenched with an equal volume 2× Laemmli buffer and analyzed by immunoblotting.