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. 2013 Mar 29;288(20):14569–14583. doi: 10.1074/jbc.M112.437392

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 6. — Sumoylation of Nrf2 in vitro. Using GST-Nrf2 fusion protein (purified as described under “Experimental Procedures”) as substrate (0.5 μg), the assays were performed with in vitro sumoylation assay kits (Active Motif), with p53 supplied in the kit as a positive control. The GST-Nrf2 fusion protein as well as products of the reaction were separated on 7% SDS-PAGE and analyzed by Western blotting using the indicated antibodies. A, representative Western blot of the purified GST-Nrf2 fusion protein. B, SUMO-1-modified p53. C and D, SUMO-1-conjugated (C) and SUMO-2-conjugated (D) Nrf2 detected with anti-Nrf2 antibody are shown in the upper panels. Lower panels show the same membranes blotted with anti-SUMO-1 and anti SUMO-2/3 antibody, respectively.