Xenbase Image ID: 129599

Figure 3. Effects of I-2, PHA-767491 and Cdk activity on DNA replication. (a) Interphase Xenopus egg extract was supplemented with demembranated sperm nuclei, [α-32P]dATP and different concentrations of PHA-767491, plus or minus I-2; after 90 min total DNA synthesis was determined. Mean and s.e.m. of three independent experiments is shown. (b) Extract was supplemented with sperm nuclei at 15 ng DNA μl−1, [α-32P]dATP and optionally with I-2. At the indicated times, total DNA synthesis was measured. Mean and s.e.m. of three independent experiments is shown. (c) Extract was supplemented with sperm nuclei at 10 ng DNA μl−1 and optionally with I-2. At the indicated times, chromatin was isolated and immunoblotted for Mcm4 and PCNA. The lower portion of the gel was stained with Coomassie to visualize histones. (d) Chromatin was prepared by incubating sperm nuclei at 20 ng DNA μl−1 in interphase extract supplemented with 50 μM PHA-767491; after 60 min chromatin was isolated and transferred to fresh extract containing [α-32P]dATP optionally supplemented with p27kip1. At different times, aliquots were taken, and total DNA synthesis was determined. Mean and s.e.m. of three independent experiments is shown. (e) ‘KIP1 chromatin’ was prepared by incubating sperm nuclei at 20 ng DNA μl−1 in interphase extract supplemented with p27kip1 and 1.2 μM I-2; after 40 min, chromatin was isolated. ‘KIP1 chromatin’ or naive sperm nuclei were then incubated in fresh extract containing [α-32P]dATP, optionally supplemented with 20 μM PHA-767491 and 1.2 μM I-2. At different times, aliquots were taken, and total DNA synthesis was determined and expressed as percentage of the amount synthesized by 90 min in control (untreated extract). Mean and s.e.m. of three independent experiments is shown. Image published in: Poh WT et al. (2014) . Creative Commons Attribution license Larger Image Printer Friendly View

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