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. 2016 Apr 10;67(11):3277–3288. doi: 10.1093/jxb/erw142

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© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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

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Fig. 6. — Ub dimer formation assay by His-UBC22. An in vitro Ub dimer formation assay was performed with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) the addition of His-UBC22. The components added in the different reactions are indicated at the top of the figure. The conjugation reactions were performed at 30 °C for 4 hours. Assay samples were subjected to SDS-PAGE. Ub and Ub dimer were detected by western blotting using an anti-Ub antibody. Free Ub and Ub dimer (Ub₂) are indicated at the right side of the figure. For the K11R mutant, no new Ub dimer formation was seen when His-UBC22 protein was added. Due to slight impurity in the recombinant K11R protein, one weak band was present which is slightly higher than the Ub dimer synthesized when His-UBC22 was added. Three experiments were performed and produced similar results.