RanGTP stabilizes MTs in anaphase and ISWI is required for MT stabilization. (A) A CSF extract was supplemented with 0.37 mg/ml rabbit IgG or anti–full-length ISWI antibody. The CSF extracts were incubated with centrosomes, anti-TPX2 antibody, and Cy3-tubulin in the presence or absence of RanQ69L at 20°C for 30 min. The samples were then cycled into anaphase and interphase by calcium addition. At each time point, the samples were fixed, spun down on coverslips, and imaged. This experiment was reproduced three times. Note that the MT stabilization assay is performed in the presence of anti-TPX2 antibody that inhibits RanGTP-dependent MT nucleation and thus keeps the number of asters constant during the assay. This is important to evaluate MT stabilization activity correctly (Yokoyama et al., 2008). Bar, 20 µm. (B) Quantification of the MT length of centrosomal asters assayed in A as described previously (Yokoyama et al., 2008). Error bars represent SD; n > 20 asters. (C) Quantification of the MT intensity of centrosomal asters assayed in A. Error bars represent SD; n > 20 asters.