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. 2013 Feb 1;41(5):3162–3172. doi: 10.1093/nar/gkt043

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Pre-RC complexes crosslinked in solution. During pre-RC formation with ATPγS, an ORC–Cdc6–Cdt1–MCM2–7 complex is formed that is labelled OCCM. During pre-RC formation with ATP, an MCM2–7 double hexamer is formed that is labelled dHex MCM2–7. The single MCM2–7 hexamer is labelled sHex MCM2–7. A small amount of single hexameric MCM2–7 can be detected in the high salt-washed pre-RC ATP owing to destabilization of the complex. (A) Analysis of crosslinked pre-RC reactions using silver staining. Purified ORC and MCM2–7 (lanes 1 and 2), crosslinked purified ORC and MCM2–7 (lanes 3 and 4), crosslinked high salt-washed pre-RC ATP (lane 5), crosslinked pre-RC ATPγS (lane 6) and low salt-washed crosslinked pre-RC ATP (lane 7) are shown. (B) Purified Cdc6, Cdt1, ORC and MCM2–7 (lanes 1, 5, 9 and 14); purified crosslinked ORC and MCM2–7 (lanes 10 and 15); crosslinked pre-RC ATP (lanes 2, 4, 6, 8, 11, 13, 16 and 18); and crosslinked pre-RC ATPγS (lanes 3, 7, 12 and 17) were analysed by Cdc6, Cdt1, Orc3 and Mcm2 Western blot.