Figure 6.

Xchk1 is modified when the DNA replication and DNA damage checkpoints are activated. (A) ³⁵S-labeled Xchk1 was incubated in interphase egg extracts containing 3,000 sperm nuclei/μl (lane 1), 3,000 UV-damaged sperm nuclei/μl (lanes 2 and 3), or 3,000 sperm nuclei/μl and 100 μg/ml aphidicolin (lanes 4 and 5) in the presence (lanes 3 and 5) or absence (lanes 1, 2, and 4) of 5 mM caffeine. After 100 min of incubation at 23°C, 2 μl of each extract was taken for SDS-PAGE and autoradiography (top). Alternatively, 50 μl of each extract was centrifuged through a sucrose solution to isolate the nuclear fraction (bottom), which was also subjected to SDS-PAGE and autoradiography. APH, aphidicolin. (B) ³⁵S-labeled Xchk1 was immunoprecipitated from interphase extracts containing 3,000 sperm nuclei/ μl and 100 μg/ml aphidicolin. The immunoprecipitates were incubated in phosphatase buffer containing no addition (lane 1), protein phosphatase 2A (lane 2), or protein phosphatase 2A and 3 μM okadaic acid (lane 3). PP2A, protein phosphatase 2A; OA, okadaic acid. (C) ³⁵S-labeled Xchk1 (lanes 1–4) and Xchk1– N135A (lanes 5–8) were incubated for 100 min in either control-depleted (lanes 1, 2, 5, and 6) or Xchk1-depleted (lanes 3, 4, 7, and 8) extracts in the presence of either 3,000 sperm nuclei/μl (lanes 1, 3, 5, and 7) or 3,000 UV-damaged sperm nuclei/μl (lanes 2, 4, 6, and 8). Nuclear fractions were isolated as described above and subjected to SDS-PAGE and autoradiography.