Figure 3.

Geminin depletion and MG132 treatment permits efficient re-replication. Interphase Xenopus extracts were optionally supplemented with MG132 and then immunodepleted with either anti-geminin or nonimmune antibodies. (A) Sperm nuclei were incubated at 10 ng DNA/μl in the extracts for 90 min; 40 μM aphidicolin was then added and the incubation continued for a further 60 min. Chromatin was isolated at either 90 or 150 min and immunoblotted for Mcm7 and Cdc6. (B) The extracts were supplemented with sperm nuclei (5 ng DNA/μl), [α-³²P]dATP and BrdUTP, and incubated for 180 min. DNA was then isolated and fractionated on CsCl equilibrium density gradients. The expected densities of heavy–heavy (HH), heavy–light (HL) and light–light (LL) DNA are indicated.