Xenbase Image ID: 124935

Figure 3. Identification of GEMC1 interacting proteinsa) WB of indicated proteins on pull-downs performed with amylose resin that was untreated (Mock), pre-bound to MBP (MBP) or to xGEMC1 fused to MBP (r-xGEMC1) and subsequently incubated in egg extract. * indicates non-specific band. b) WB of TopBP1 on pull-downs performed with amylose resin that was untreated (Mock), pre-bound to MBP (MBP) or to GEMC1 fused to MBP (r-GEMC1) and incubated with extract (+ Extract) or with recombinant TopBP1. c) Chromatin binding of the indicated proteins at different times after addition of sperm nuclei (3000 n/μl) to egg extracts that were mock (Mock) or TopBP1 (ΔTopBP1) depleted. d) Chromatin binding of the indicated proteins at different times after addition of sperm nuclei (3000 n/μl) to interphase egg extracts that were mock (Mock) or Orc1 depleted (ΔORC1). e) Autoradiograph of 35S-labelled xGEMC1 (35S-GEMC1) or 35S-labelled xGEMC1 mutated in the Cyclin binding site (35S-xGEMC1-ANA) produced in reticulocyte lysates and incubated with buffer (−) or recombinant Cdk2-CyclinE complex (Cdk2-E). f) Amount of replicated DNA measured by quantifying α32P-dATP incorporation over time using TCA precipitation (see methods) at different times after the addition of 1000 or 6000 nuclei/μl to egg extracts supplemented with 300 ng/μl recombinant xGEMC1 (r-xGEMC1), 300 ng-μl xGEMC1-8ST-D (r-xGEMC1-8ST-D) carrying the serine and threonine residues phosphorylated by Cdk2 mutated to aspartate, 300 ng/μl xGEMC1-8ST-A protein (r-xGEMC1-8ST-A) with the same residues mutated to alanine, with 3 mM caffeine (Caff) or with 3 mM caffeine in the presence of 300 ng/μl xGEMC1-8ST-D protein (xGEMC1-8ST-D + Caff). The graphs show data from one representative experiment. xGEMC1 proteins used in these experiments were defective for the Cyclin-binding domain (R198NL to A198NA mutation) to avoid titration of endogenous Cdk2-Cyclin E complex. g) Cdc45 chromatin binding. Egg extracts were supplemented with buffer (Buffer) or 300 ng/μl xGEMC1-8ST-D mutant protein (r-xGEMC1-8ST-D). Orc1 was used as loading control. h) WB of TopBP1 (top panel) on pull-downs performed with amylose resin that was pre-bound to MBP (MBP), to MBP fused to wild type xGEMC1 (r-xGEMC1), to aspartate (r-xGEMC1-8ST-D) or to alanine substituted xGEMC1 (xGEMC1-8ST-A) (bottom panel, coumassie) and incubated with recombinant TopBP1. See Supplementary Fig 6 for uncropped gels shown in this figure. Image published in: Balestrini A et al. (2010) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View

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