Ubiquitination Assay for SUMO-2 Chains

Ubiquitination assays were carried out as previously described^{12 (link)}. The following components were mixed together and incubated at room temperature: 0.1 μM E1, 2.5 μM Ubc13, 2.5 μM Ube2V2, 0.55 μM RNF4, 5.5 μM Ub~4xSUMO-2 or 4xSUMO-2, 20 μM Ub, 3 mM ATP, 5 mM MgCl₂, 50 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 0.1 % NP40. The reaction was stopped with SDS-PAGE loading buffer and analyzed by SDS-PAGE. Gels were stained with Coomassie Blue. Time points were taken at 0, 2, 5, 10, 20, 40, 60 and 100 min. The zero time point was taken before the addition of ATP. Reactions were also analyzed by western blotting with anti-ubiquitin antibody (Dako).