Figure 5.

Response of Xenopus egg extracts to poly(dT)₄₀ in the presence and absence of Xcds1. (A) A simple DNA homopolymer delays mitosis. Poly(dT)₄₀ (○, □) or poly(dG)₄₀ (●, ▴) was added to the extracts at a final concentration of 50 ng/μl in the presence (□, ▴) or absence (○, ●) of 5 mM caffeine. The extracts were activated with CaCl₂ before the addition of DNA. Sperm nuclei (200 nuclei/μl) were added to the extracts to monitor the timing of nuclear envelope breakdown (NEB) by microscopy. (B) Removal of Xcds1 and Xchk1 proteins from egg extracts by immunodepletion. M-phase extract (100 μl) was incubated with a mixture of anti-Xcds1 antibodies (20 μg) and anti-Xchk1 antibodies (10 μg) bound to Affiprep protein A beads for 50 min at 4°C with constant rocking. Protein A beads were removed by centrifugation. A second round of depletion was then performed to ensure that both Xcds1 and Xchk1 were completely removed, which was assessed by immunoblotting. As a control, M-phase extracts underwent the same procedure with nonspecific rabbit IgG. For depletion of Xcds1 alone, anti-Xchk1 antibodies were omitted. (C) Depletion of Xcds1 does not diminish the mitotic delay caused by poly(dT)₄₀. Sperm nuclei (500 nuclei/μl) (●, ▴) or both sperm nuclei (500 nuclei/μl) and poly(dT)₄₀ (50 ng/μl) (○, □) were added to Xcds1-depleted (○, ●) or mock-depleted (□, ▴) extracts. (D) Same as C except that both Xcds1 and Xchk1 were depleted.