Figure 4. CDK1/cyclin B upregulates SUMOylation by increasing thioester bond formation between phosphorylated Ubc9 and SUMO1 but not between SAE1/SAE2 and SUMO1.

(A) To examine the thioester bond formation between His₆-SUMO1 and SAE1/His₆-SAE2, purified His₆-SUMO1 and SAE1/His₆-SAE2 were incubated with various concentrations of CDK1/cyclin B (1.5 nM, 3 nM and 6 nM) in the presence or absence of ATP. Reaction mixtures were run on a 10% SDS-PAGE and immunoblotted with anti-SAE1/SAE2 antibody. (B) Ubc9 thioester conjugation assay was performed by incubating purified His₆-Ubc9, SAE1/His₆-SAE2 and His₆-SUMO1 with various concentrations of CDK1/cyclin B (1.2 nM, 3 nM and 6 nM) in the presence of ATP for 60 min at 37°C. Samples were then resolved on a 15% SDS-PAGE and probed with anti-Ubc9 antibody. (C) Ubc9 thioester conjugation assay in was performed similar as in (B), but at the end of the reaction, 200 mM of DTT was added to samples for overnight at 40°C. The reduced samples were run on a 15% SDS-PAGE, followed by immunoblotting with anti-Ubc9 antibody. Iso, isopeptide bond; Thiol, thioester bond.