Ube2s

In vitro ubiquitination assays were performed as previously described with slight modifications (Oh et al., 2020) (link). The assays were carried out in a 20 μL reaction volume, and components of complete reactions included: 0.5 μL of 5 μM UBE1 (125 nM final, R&D Systems), 1 μL of 10 μM UBE2S (0.5 μM final, R&D Systems), 1 μL of 10 μM UBE2C (0.5 μM final, R&D Systems), 1 μL of 10 mg/mL His-TEV-ubiquitin (0.5 μg/μL final, prepared as described below), 2 µL of 0.5 mg/mL securin (0.5 μg/μL final, Abcam), 1 µL of 100 μM DTT, 1.5 μL of energy mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl2, 2 mM EGTA, pH to 7.5), 2 µL of 10X assay buffer (500 mM NaCl, 100 mM MgCl2 and 250 mM Tris pH 7.5), 5 μL of PAC/C coupled resin and 5 µL of 1X PBS. For control reactions that lacked a certain component, the mixture was supplemented accordingly with 1X PBS. Reactions were performed at 30 ºC with shaking for 30 min and stopped by addition of 6X Laemmli Buffer and incubation at 95 ºC for 5 min. Samples were analyzed by SDS-PAGE and Western blot.