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. 2011 Sep 1;2(5):455–464. doi: 10.4161/nucl.2.5.17859

Figure 4.

Figure 4

Nature of licensing inhibitor in M phase and S phase hamster extracts. (A and C) Step 1 of the licensing assay was performed with highspeed Xenopus extract mixed with buffer alone (grey) or with M phase (A; ratio of 1:1) or S phase (C; ratio of 3:1) CHO extract (white). Prior to mixture, buffer or CHO extracts were supplemented with the Cdk inhibitor 6-DMAP (3 mM), a geminin-neutralizing fragment of Cdt1 (amino acids 193–447; Con7, 5 ng/µl), or both inhibitors simultaneously. DNA synthesis in Step 2 is expressed as a percentage of that obtained with buffer alone. Shown are the means of three experiments with independent extract batches and error bars show standard deviation. (B and D) Xenopus sperm chromatin was incubated with high-speed Xenopus extract mixed with buffer (+Buffer) or hamster M-phase (B) or S-phase (D) extract supplemented with inhibitors as in (A and C) except that Roscovitine (rosco, 40 µM) was substituted for 6-DMAP as a more potent and specific inhibitor of Cdk activity. Following the 25 min Step 1 incubation period, sperm chromatin was isolated and subjected to immuno-blotting to evaluate Mcm protein loading.