Figure 4

XDbf4 chromatin association is independent of a pre-replication complex. (A) Sperm nuclei were incubated for 30 min in interphase extracts minus or plus added Cip1 (extract+Cip1) or geminin (extract+geminin). Chromatin was isolated, subjected to SDS-PAGE and immunoblotted for XOrc1, XCdc6, XMcm7, XDbf4 and XCdc7. A control lacking added sperm is also shown. (B) Sperm nuclei were incubated at 10 ng/μl in interphase Xenopus egg extract in the presence of geminin. At the indicated times, chromatin was isolated and immunoblotted with antibodies specific for XOrc1, XCdc6, XMcm7, XDbf4 and XCdc7. Equivalent exposures to the time course of Fig 3 are shown. (C) Sperm nuclei were incubated for 40 min in interphase extracts previously immunodepleted with antibodies against XOrc1 (XOrc1-), XCdc6 (XCdc6-), and XMcm7 (XMcm7-), or with non-immune antibodies (NI-). The incubations also were performed in the presence of the phosphatase inhibitor microcystin (μC) or with a control lacking added sperm. Chromatin was then isolated and immunoblotted for XOrc1, XCdc6, XMcm7 and XCdc7. A Coomassie-stained image of the gel containing the histones is shown as loading control. (D) Sperm nuclei were incubated for 1 hr in extract supplemented with geminin and/or 5 mM caffeine. Chromatin was then isolated and immunoblotted with antibodies against XDbf4 and XCdc7. In parallel, nuclei were isolated and immunoblotted for phospho-Ser³⁴⁴-Chk1.