Figure 1.

Characterization of mitotic kinases in Xenopus egg extracts. (A) Sperm chromatin was incubated with interphase low-speed supernatants (LSS) to assemble interphase nuclei; a half volume of CSF LSS was added to trigger entry into mitosis. Chromatin fractions were isolated at the times indicated (lanes 3–13) and analyzed by immunoblotting with antibodies specific to Xenopus Rad21 (XRAD21, panel 1), XCAP-H (panel 2), Plx1 (panel 3), Xenopus aurora B (XAUB, panel 4), Xenopus INCENP (XINC, panel 5), Xenopus survivin/BIR1 (XBIR1, panel 6), and histone H3 phosphorylated at serine 10 (H3-P, panel 7). An aliquot of extract (lane 1) and a sample from a mock assembly reaction without sperm chromatin (lane 2) were also included. (B) Immunoprecipitations were performed from interphase high-speed supernatants (HSS) using affinity-purified antibodies against INCENP (lanes 1,2), aurora B (lanes 3,4), and BIR1 (lanes 5,6). The corresponding antigen peptides were added at 0.4 mg/mL to demonstrate the specificity of these reactions (lanes 2,4,6). The immunoprecipitates were analyzed by immunoblotting with the antibodies indicated. Alternatively, the immunoprecipitates obtained with the anti-XINC antibody in the absence (lane 7) or presence (lane 8) of XINC peptide were analyzed by silver staining. The positions of the three polypeptides as well as the IgG heavy chains (IgG HC) and light chains (IgG LC) are indicated. (C) An aliquot of an interphase HSS was overlaid onto a 5%–20% sucrose density gradient and spun at 36,000 rpm for 14 h in a SW50.1 rotor (Beckman). Fractions were taken manually and analyzed by immunoblotting. Aurora B, INCENP, and survivin/BIR1 comigrate in the indicated 11S peak.