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. 2008 Feb 28;27(6):876–885. doi: 10.1038/emboj.2008.29

Figure 5.

Figure 5

Plx1 interacts with the Mcm complex through its Polo box domain. (A) Immunodepletion of Tipin. Extracts were mock depleted (Mock) or Tipin depleted using anti-Tipin antibodies (ΔTipin). The cytosol was then subjected to western blot with anti-Tipin antibodies. (B) Plx1 and Claspin binding in Tipin-depleted extracts. Sperm nuclei were incubated in mock-depleted (Mock) or Tipin-depleted (ΔTipin) extracts in the presence of 2.5 μM aphidicolin (APH). Chromatin was isolated at the indicated time points and probed with anti-Plx1 and anti-Claspin antibodies. (C) Co-immunoprecipitation of Plx1 and Mcm7. Extracts that were untreated (−) or treated with 50 ng/μl pA/pT (+) were immunoprecipitated with pre-immune (Pre-Imm) or anti-Plx1 (Anti-Plx1) antibodies. Western blot was performed using anti-Mcm7 and anti-Plx1 antibodies. (D) Pull-down with GST–Polo box and MALDI-TOF analysis. Interphase extracts were incubated with GST–agarose beads or agarose beads with GST fused to the Polo box derived from human Plk1. After pull-down, proteins were consecutively eluted using 3 mg/ml of phosphopeptide solution (first elution=E1, second elution=E2, see Supplementary data for the sequence of the peptide and the Polo box) and subjected to MALDI-TOF analysis. Band 1 corresponds to Mcm2, band 2 to Mcm6, band 3 to Mcm4 and band 4 to Mcm7. The GST fusion protein containing the Polo box from Plk1 is shown at the bottom. (E) GST–Polo box pull-down of nuclear proteins probed with anti-Mcm7 antibodies (GST–Polo box). The extract used for the pull-down was supplemented with nuclei and buffer (Buff), 2.5 μM aphidicolin (APH) or 2.5 μM aphidicolin and 5 mM caffeine (Caff), as indicated. The total amount of unbound Mcm7 extracted from nuclei was loaded as a control (Input). (F) Effects of Mcm complex depletion on Plx1 binding to chromatin. The extract was untreated (C), mock depleted (M) or Mcm depleted (ΔMCM) using anti-Mcm3 antibodies and incubated with sperm nuclei for 60 min in the presence of 2.5 μM APH. Cytosol and chromatin were then subjected to western blot with anti-Mcm7 and anti-Plx1 antibodies.