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. 2012 Aug 13;40(20):10394–10407. doi: 10.1093/nar/gks763

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© The Author(s) 2012. 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 4. — Preferential utilization of UBC13∼Ub as Ub acceptor by HLTF. (A) Time-course of chain formation using the minimal set of factors. Reaction mixtures containing mpssDNA, E1, MMS2–UBC13 and Ub were pre-incubated for 10 min and then the reactions were started by adding HLTF and incubated for the indicated times. Reaction products were treated with or without a reducing agent and then analyzed by immunoblot using an anti-Ub antibody. (B) UBC13∼Ub_n competition assays. Reactions in the presence of a 10-fold excess of Ub (10×) were compared to those under standard reaction conditions (1×) with the minimal set of factors. The molecular ratios of UBC13 to Ub are shown in parentheses. Reaction mixtures were pre-incubated without HLTF for 1 min and then the reactions were started by adding HLTF and incubated for the indicated times. Reaction products untreated by a reducing agent were analyzed by immunoblot using an anti-UBC13 antibody. Presumed dimers of UBC13 linked via S–S bond formation during sample preparation are indicated by an arrowhead.