Fig. 2.

A strategy for discerning SUMOylation dynamics during replication stress. A, Cartoon depicting the strategy to study SUMOylation dynamics during replication stress. U2OS cells expressing His10-SUMO-2 were treated with 2 mm Hydroxyurea (HU) for 2 h or 24 h to induce DNA replication fork stalling and double strand breaks, respectively. Parental U2OS cells and U2OS cells expressing His10-SUMO-2 were mock treated as negative controls. SUMO-2 target proteins were purified by Ni-NTA purification. To study SUMO-2 targets that dynamically respond to replication stress, five biological replicates were performed. B, Purification of His10-SUMO-2 conjugates via NTA purification was confirmed by immunoblotting. Whole cell extracts and SUMO-2 purified proteins of the differently treated cells were run on 4–12% Bis-Tris polyacrylamide gels and levels of His10-SUMO-2 conjugates were compared by immunoblotting using anti-SUMO-2/3 antibody.