Fig. 3. Requirement of Cut5 for DNA replication. (A) Immunodepletion of Cut5 from S-phase egg extracts. The egg extracts were treated with control and anti-Cut5 antibodies bound to a protein A matrix. Aliquots of mock-depleted (mock) and Cut5-depleted extracts with (ΔCut5+ rec.Cut5) or without (ΔCut5) 5 ng/µl of recombinant Cut5 were resolved by SDS–PAGE, then immunoblotted with antibodies against the proteins indicated in the figure. (B) Replication activity of Cut5- depleted extracts. Sperm nuclei were incubated in the treated egg extracts for the indicated times at 23°C. Replication activity was monitored as the incorporation of [α-³²P]dCTP into sperm DNA. Replication products were subjected to agarose gel electrophoresis followed by autoradiography. (C) Relative intensities of autoradiography were analyzed and plotted against time, taking the highest value of the mock-depleted extracts as 100%.