Generation of recombinant GST-OTUB2 protein

In brief, a single plasmid-transformed E. coli BL21 (DE3) colony was inoculated into 20 ml of LB medium supplemented with 100 µg/ml ampicillin and grown overnight at 37 °C, 200 r.p.m. The overnight culture was transferred to 1 L of LB medium with ampicillin and grown at 37 °C, 200 r.p.m., for ~3–4 h until OD₆₀₀ reached 0.6. The culture was added with 1 mM IPTG and further incubated at 18 °C, 200 r.p.m., for 12 h. E. coli cells were harvested by centrifugation and resuspended in 30 ml PBS (containing 5 mM DTT). Cell suspensions were homogenized with a SONICS Vibra–Cell VCX-800 ultrasonic processor at 4 °C for 12 min and then centrifuged at 25,000 g at 4 °C for 30 min. The GST-OTUB2 protein in clarified lysate was purified using a glutathione sepharose 4B column (GE Healthcare, 17075601). GST protein elution was conducted with 5 mM reduced GSH in 50 mM Tris–HCl buffer (pH 8.0). Purified protein was concentrated and dialyzed in PBS overnight at 4 °C. Protein concentration was measured using a BCA Protein Assay Kit (Pierce, 23225), diluted to 1 mg/ml and stored at −20 °C.