SUMOylation on mitotic chromosomes regulates Haspin binding and H3T3 phosphorylation. (A) Mitotic replicated chromosomes prepared as in Fig. 1 A with (Cont.) or without (+dnUbc9) mitotic SUMOylation. Isolated chromosomes were analyzed by immunoblotting with indicated antibodies. Histone H3 was used as a loading control for the mitotic replicated chromosomes. (B) Quantification of Haspin and H3T3p levels on the mitotic replicated chromosomes, as seen in A, relative to levels of Cont. chromosomes from three independent experiments (n = 3) with levels normalized to histone H3 levels. Error bar represents SD. *, P < 0.05 (Student’s t test). (C) Mitotic replicated chromosomes prepared from CSF XEEs with (Cont.) or without (+dnUbc9) mitotic SUMOylation were subjected to immunofluorescence staining with antibodies as indicated with Hoechst 33342. (D) Quantification of H3T3p signal intensity at the mitotic centromeres, as seen in C, relative to signal intensities of Cont. centromeres from three independent experiments (n = 3, 40 centromeres per n) with levels normalized to CENP-A. Error bar represents SD. ***, P < 0.001 (Student’s t test).