Aurora B kinase activity assay

Cell lysates from post-treatment samples were incubated with a primary anti-Aurora B antibody (Abcam) overnight at 4°C. Twenty microliters of protein A/G agarose magnetic beads (Thermal Scientific) were added into 500 μL cell lysate and incubated for 2 hours at 4°C. The pellet was washed three times with a pH 7.5 kinase assay buffer (20 mM Tris, 10 mM MgCl₂, 5 mM glycerophosphate, 0.1 mg/mL BSA, 2 mM dithiothreitol), and resuspended in 30 μL kinase buffer supplemented with 200 μM ATP and 2 μg histone H3.1 protein (Biolabs Inc.). The mixture was incubated for 30 min at 30°C to allow protein phosphorylation, and terminated with a SDS sample buffer before applying for Western blot analysis for histone H3.