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. 2010 Nov 5;9(21):4364–4369. doi: 10.4161/cc.9.21.13632

Figure 3.

Figure 3

Recovery from the DNA damage response in extracts. (A) The cell-free checkpoint recovery system. Metaphase II-arrested CSF extracts were released into interphase for 30 min by addition of Ca++. Magnetic beads conjugated with dA-dT were added to 20 µg/ml and incubated in the extract for 30 min then removed with a magnet as described in Materials and Methods to mimic the completion of DNA repair. (B) Analysis of dA-dT beads removed from extracts. As in (A), extracts before and after dA-dT removal, as well as the removed beads were analyzed by western blotting using the indicated antibodies. Lane 1, extract before dA-dT removal; lane 2, extract after dA-dT removal; lane 3, dA-dT beads removed from lane 1. (C) The removal of dA-dT from extracts depicted in (A) enables deactivation of DNA damage signaling and re-entry into mitosis, as judged by western blotting of Smc1, Chk1, Cdc2 and Cdc27. Control extracts without removal of dA-dT (left) sustained the checkpoint arrest.