ISWI localizes in interphasic nuclei and on mitotic spindles in Xenopus culture cells and egg extracts. (A) Immunoblot of Xenopus XL177 cell lysate and CSF extract with 1 µg/ml of affinity-purified ISWI antibodies against full-length (1–1046 aa), N-terminal (1–400 aa), or C-terminal (746–1046 aa). (B) Localization of endogenous ISWI in Xenopus XL177 cells. Cells were fixed in cold methanol. ISWI was stained with a mixture of the three ISWI antibodies (total 3 µg/ml of each 1 µg/ml) and subsequently with Alexa 488–labeled anti–rabbit IgG (green). Tubulin was stained with anti-tubulin and Alexa 568–labeled anti–mouse IgG (red). DNA was stained with Hoechst 33342 (blue). Bar, 10 µm. (C) Localization of recombinant ISWI in Xenopus egg extracts. A CSF extract was immunodepleted with anti–N-terminal ISWI antibody or rabbit IgG, and 1.0 µM GFP-ISWI and GFP control (green) were added back to the extract, respectively. Each extract was supplemented with sperm nuclei and Cy3-labeled tubulin (red), and sent to interphase by adding calcium and incubating at 20°C for 90 min. The samples were cycled into metaphase by adding ISWI- or mock-depleted CSF extract and incubating at 20°C for 60 min, respectively. The samples were further cycled to anaphase by calcium addition and 30 min incubation. At each step, small aliquots were fixed, spun down onto coverslips, and stained with Hoechst (blue). Bar, 20 µm. (D) Endogenous ISWI localizes on mitotic spindles in egg extracts. Cycled sperm spindles were fixed, spun down on coverslips, and stained with or without 1 µg/ml anti–C-terminal ISWI antibody. The coverslips were subsequently stained by Alexa 488–labeled anti–rabbit IgG (green). MTs are red and DNA is blue. Bar, 20 µm.