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XB-IMG-170420

Xenbase Image ID: 170420

Figure 5. Effect of RL5a on Licensing-Defective ExtractsSperm nuclei were incubated in Xenopus egg extract that had optionally been depleted of MCM3 or Cdc6 or which had been supplemented with geminin and which were further supplemented with 200 μM RL5a or DMSO. After 20 min, chromatin was isolated in 50, 100, or 200 mM KCl and then immunoblotted for ORC (Orc1 subunit) or Cdc6.(A and B) A representative series of blots is shown in (A). Blots from at least three separate experiments were quantified for the amount of chromatin-bound ORC (Orc1 subunit) and Cdc6. The mean signal ± SEM, relative to control chromatin isolated in 50 mM KCl, is plotted in (B).(C) Plasmid DNA (pET28) was incubated in a partially purified fraction of ORC supplemented with 2.5 mM ATP and either 25, 50, 100, 150, or 200 μM RL5a or DMSO. After 30 min, DNA was isolated in 100 mM KCl and then immunoblotted for Orc1 and Orc2.(D) Orc1 and Orc2 plasmid DNA association in (C) was quantified relative to recovered DNA and expressed in relation to control (mean ± SEM, n ≥ 5).See also Figure S5.

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