Inhibition of SUMOylation reduces Aurora B kinase on mitotic chromosomes. (A) Schematic method for the preparation of mitotic replicated chromosomes from XEEs. (B) Mitotic replicated chromosomes isolated as in A with (+dnUbc9) or without (control [Cont.]) dnUbc9 were subjected to immunoblotting. Histone H3 was used for the loading control for the mitotic chromosomes. (C) Quantification of Aurora B and Aurora B T248p levels on the mitotic chromosome, as seen in B, relative to levels of Cont. chromosomes from three independent experiments (n = 3) with levels normalized to histone H3 levels. Error bars represent SD. *, P < 0.05 (Student’s t test). (D) Mitotic replicated chromosomes prepared as in A with or without dnUbc9 (Cont.) were subjected to immunofluorescence staining with antibodies as indicated with Hoechst 33342. Bar, 10 µm. (E) Quantification of the Aurora B signal intensity at mitotic centromeres, as seen in D, relative to signal intensities of Cont. centromeres from three independent experiments (n = 3, 50 centromeres per n) with levels normalized to CENP-A signal. Error bars represent SD. *, P < 0.05 (Student’s t test).