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. 2013 May 1;24(9):1343–1353. doi: 10.1091/mbc.E13-01-0025

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© 2013 Lee and Dunphy. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — MRN and Rad17 regulate activation of Chk1 in an additive manner. (A) Mock-treated (lanes 1 and 2) or Nbs1-depleted (lanes 3 and 4) extracts were incubated in the absence (lane 1) or presence (lanes 2–4) of APH. Recombinant MRN was added back to the incubation shown in lane 4. Chromatin fractions were prepared and immunoblotted for the indicated proteins. (B) Quantitation of binding of proteins to chromatin in mock-treated extracts incubated in the absence (1) or presence (2) of APH and in Nbs1-depleted extracts incubated in the presence of APH (3). Values, compiled from two independent experiments, are expressed relative to mock-treated, APH-containing extracts. (C) Egg extracts were subjected to an immunodepletion procedure with control antibodies (lane 1), anti-Rad17 antibodies (lane 2), anti-Nbs1 antibodies (lane 3), or both anti-Rad17 and anti-Nbs1 antibodies (lane 4). Extracts were immunoblotted for the indicated proteins. (D) Extracts from (C) were incubated in the absence (lane 1) or presence (lanes 2–5) of APH. Chromatin fractions were immunoblotted for the indicated proteins. (E) Extracts from (C) were incubated with [³⁵S]Chk1 in the absence (lane 1) or presence (lanes 2–5) of APH. Nuclear fractions from the extracts were processed for phosphorimaging to detect [³⁵S]Chk1 (second panel from top) or for immunoblotting with antibodies that detect pSer-344 of Chk1, pSer-864 of Claspin, Claspin, pSer-1131 of TopBP1, and TopBP1, as indicated in the remaining panels.