Figure 1.

Effects of Plx1 depletion on DNA replication. (A) Immunodepletion of Plx1. Western blot analysis with anti-Plx1 antibodies of control (C) mock-depleted (M), Plx1-depleted (ΔPlx1) and Plx1-depleted extracts reconstituted with 6 ng/μl recombinant Plx1 (+Plx1). (B) Replication assay in unchallenged extracts. Extracts supplemented with 2000 nuclei/μl were untreated (CTRL), mock depleted (Mock), Plx1 depleted (ΔPlx1) and Plx1 depleted and supplemented with recombinant Plx1 (+Plx1). DNA was replicated in the presence of [α-³²P]dATP for 120 min, extracted and separated on an agarose gel. The gel shown represents a typical result. (C) Chromatin binding of Mcm7. Sperm nuclei were replicated in untreated extracts (CTRL) and extracts supplemented with a low concentration (40 nM) of geminin (GEM). Chromatin was isolated at the indicated time points and separated on SDS–PAGE. Western blots were performed with antibodies against human Mcm7. (D) Effects of geminin on DNA replication. Extracts were supplemented with increasing concentrations of geminin (40, 60 and 120 nM). (E) Phosphorylation of Chk1 serine 345 (p-CHK1) in extracts supplemented with 7000 nuclei/μl. Extracts were untreated (−) or supplemented (+) with 40 nM geminin (GEM). Western blot was performed with anti-phospho-serine 345 of human Chk1 (p-Chk1) and anti-Chk1. Chk1 phosphorylation induced by geminin was detected with ECL plus reagent (see Materials and methods). (F) Replication assay in extracts supplemented with 40 nM geminin (GEM) in the presence of 7000 nuclei/μl. Extracts were untreated (CTRL), mock depleted (Mock), Plx1 depleted (ΔPlx1) and Plx1 depleted and supplemented with recombinant Plx1 (Plx1-dep+Plx1). The gel shown represents a typical result.