ISWI is required to stabilize spindle MTs in anaphase independently of chromosome segregation. (A) Immunodepletion of ISWI. A CSF extract was immunodepleted using rabbit IgG or anti–N-terminal ISWI antibodies. Each extract (1 µl) was immunoblotted by anti–N-terminal ISWI antibodies. (B) Determination of endogenous concentration of ISWI in Xenopus CSF extracts. A CSF extract (0.3 µl and 1.0 µl) and recombinant ISWI (1.0 µl from described concentrations) was loaded on an SDS-PAGE gel and immunoblotted with anti–N-terminal ISWI antibodies. We estimate that the endogenous concentration is ∼200 nM. (C) Spindle MTs disappear at anaphase onset in the absence of ISWI that is restored by recombinant ISWI. The mock or ISWI-depleted extracts were supplemented with DNA beads and Cy3-tubulin, and cycled into interphase and then back into mitosis. The metaphase extracts containing DNA bead spindles were sent to anaphase by calcium addition. At each time point, small aliquots of the samples were fixed on coverslips by squashing. Recombinant ISWI (1 µM) was added back to ISWI-depleted extracts before the reactions. This experiment was reproduced four times. Bar, 20 µm. (D) Quantification of the MT amounts around DNA beads assayed in C. MT intensity around DNA bead clusters containing 10–40 beads was quantified using a macro. Note that the clusters containing 10–40 beads mostly formed bipolar spindles in metaphase extracts, whereas the clusters with less than 10 beads did not nucleate MTs and the clusters with more than 40 beads formed multipolar spindles. Error bars represent SD. n > 40 structures; n > 2 experiments. (E) Cell cycle progression is normal in ISWI-depleted extract. Histone H1 kinase activity was assayed in metaphase (m), and 10, 20, 30, 40, and 60 min after calcium addition.