Fig. 5. Requirement of Cut5 for the loading of Cdc45 onto chromatin. (A) Sperm nuclei were incubated in mock-, Cut5- and Cdc45-depleted egg extracts for 40 min at 23°C. For each extract, 1.5 µl depleted extract and the isolated chromatin fractions (chromatin) from 50 µl extracts were resolved with SDS–PAGE and immunoblotted. (B) Co-immunoprecipitation of Cdc45 with Cut5 on replicating chromatin. Sperm nuclei were incubated in S-phase egg extract in the presence of 50 µg/ml aphidicolin for 50 min at 23°C. Chromatin DNA was fragmented by sonication and the fragmented chromatin fractions were clarified by centrifugation. The egg extracts and the fragmented chromatin fractions (chromatin-IP) were immunoprecipitated with antibodies as indicated, then the immunoprecipitates were resolved with SDS–PAGE and immunoblotted.