Condensin II prevents eviction of CENP-A nucleosomes from centromeres. (A) Left, subunit composition of the two condensin complexes. Right, immunoblot analysis of increasing amounts of a mock-depleted extract (expressed as percentage of a 1.5-µl aliquot) and extracts depleted of condensin I, condensin II, or both with the indicated antibodies. RbAp48 is shown as loading control. (B) Sperm chromatin was added to mock-depleted (top) and condensin-depleted (bottom) CSF extracts and incubated for 90 min. To serve as reference, sperm chromatin was added to an undepleted CSF extract, incubated for 40 min, and then calcium was added and incubation continued for 90 min. The latter samples (replicated interphase nuclei, as evidenced by the incorporation of biotin dUTP) were mixed with the former ones (mitotic chromosomes), centrifuged over coverslips, and stained with anti-CENP-A (green), DAPI (red), and streptavidin (cyan). Representative images of the photographed pairs are shown, with corresponding blown-up images of each individual centromere. (C) CENP-A signals in chromosomes assembled in depleted CSF extracts were measured in comparison with a reference sample, as described in B. The resulting numbers are expressed and plotted as fold variation relative to the average value obtained for the mock-depleted extract, arbitrarily set to 1 (red dotted line). Data from at least two independent experiments are shown for the depletion of condensins and from a single experiment in the case of CENP-A and xHJURP depletions. (D) Immunoblot analysis of chromatin fractions from assembly reactions in mock- and condensin II–depleted extracts. An aliquot of extract (lane 1) and a chromatin fraction from a mock assembly reaction with no sperm (lane 2) were also analyzed.