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. 2009 Oct 1;20(19):4246–4255. doi: 10.1091/mbc.E09-05-0378

Figure 2.

Figure 2.

CENP-C assembly in the Xenopus egg extract system. (A) CENP-C is not retained in Xenopus sperm chromatin. After decondensation with Xenopus Nap-1, Xenopus sperm nuclei show staining for CENP-A but not CENP-C, whereas Xenopus somatic cell nuclei stain for both CENP-A and -C. Bar, 5 μm. (B) Immunoblot of Xenopus sperm preparation and XTC cell nuclei, indicating the levels of CENP-A and -C in the sperm preparation and in somatic nuclei. Crude Xenopus egg extract (Extract), nuclei from the sperm preparation (Sperm prep, 5 × 106 total nuclei with 3.95 × 105 contaminating somatic nuclei), and XTC cell nuclei corresponding to the number of contaminating somatic nuclei in the sperm preparation (Somatic nuclei control, 3.95 × 105 nuclei from XTC cells). (C) CENP-C is ∼10 nM in CSF egg extract by immunoblot. The level of CENP-C in crude Xenopus egg extract (Extract) was compared by Western blotting to serial dilutions of a purified GST fusion of CENP-C (AA207–296) as a standard. A Coomassie-stained gel of the purified GST fusion is shown on the right. (D) CENP-C assembles onto sperm chromatin centromeres after incubation of sperm in either metaphase arrested CSF (top) or interphase (bottom) egg extract. Bar, 5 μm.