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Fig. 8. ATR is necessary for the aphidicolin-induced intra-S-phase checkpoint. (A,B) Sperm nuclei were incubated at 15 ng DNA µl^-1 in extract supplemented with [-³²P]dATP, and various concentrations of aphidicolin, plus or minus 5 mM caffeine and (A) an antibody neutralizing the ATR kinase (-X-ATR) or (B) 800 nM wortmannin. At 120 minutes total DNA synthesis was measured. As control, aphidicolin was substituted with DMSO. (C,D) Sperm nuclei were incubated at 15 ng DNA µl^-1 in extract supplemented with 0 µM, 15 µM or 120 µM aphidicolin, plus or minus 800 nM wortmannin or 5 mM caffeine. (C) At 50 minutes (mid-S-phase), chromatin was isolated and immunoblotted for Cdc45 and PCNA. (D) At 60 minutes, intact nuclei were isolated and immunoblotted for phospho-Ser344-Chk1. (E) Sperm nuclei were incubated at 15 ng DNA µl^-1 in extract supplemented with 12 µM aphidicolin plus or minus 5 mM caffeine or 5 µM DBH. At 120 minutes, total DNA synthesis was measured. (F,G) Extract was immunodepleted with anti-Chk1 antibodies. (F) Immunoblotting of whole nuclei assembled in either untreated extract, mock depleted or Chk1-depleted extracted showed the removal of Chk1. `?' indicates an unknown cross-reacting protein. (G) Sperm nuclei were incubated at 15 ng DNA µl^-1 in Chk1-depleted extract, non-immune-depleted extract or untreated extract, all supplemented with [-³²P]dATP and combinations of 10 µM aphidicolin and/or 5 mM caffeine. At 120 minutes, total DNA synthesis was measured.

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