Stimulation of chromosomal binding of condensin I by OA in interphase extracts

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Analysis of the role of Aurora B on the chromosomal targeting of condensin I"</p><p></p><p>Nucleic Acids Research 2007;35(7):2403-2412.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874644.</p><p>© 2007 The Author(s)</p> Sperm nuclei were incubated with mitotic extract (lanes 1–3), interphase extract (lanes 4–6), interphase extract supplemented with 1.2 (lanes 7–9), 3.6 (lanes 10–12) or 12 μM (lanes 13–15) of OA at 22°C for 2 h. Chromatin-bound proteins were dissolved with SDS-PAGE sample buffer, and 12.5 (lanes 1, 4, 7, 10 and 13), 25 (lanes 2, 5, 8, 11 and 14) and 50% (lanes 3, 6, 9, 12 and 15) of each sample were separated by SDS–PAGE, and immunoblotted with anti-phospho H3. Samples were prepared as described in (A), and 6.25% (lane 1), 12.5% (lanes 2, 4, 7, 10 and 13), 25% (lanes 3, 5, 8, 11 and 14) and 50% (lanes 6, 9, 12 and 15) of samples were blotted using anti-XCAP-E (upper), and anti-XCAP-G (lower) antibodies. Sperm chromatin was assembled in the mitotic extract (a, d), interphase extract (b, e, g), and interphase extract supplemented with 3.6 μM OA (c, f, h). Samples were fixed and stained with Hoechst (a, b, c; first low), and anti-XCAP-H antibody (d, e, f; second low, short exposure: g, h; third low, long exposure). Bar, 10 μm. () Sperm chromatin was assembled in the mitotic extract (a, c, e) or interphase extract supplemented with 3.6 μM OA (b, d, f). After assembly, the reaction mixtures were supplemented with the indicated extra concentration of KCl at 22°C for 20 min, fixed, and stained with Hoechst. Bar, 10 μm