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. 2012 Dec 4;288(3):1785–1794. doi: 10.1074/jbc.M112.439760

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

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FIGURE 4. — Arkadia is polyubiquitinated through K27- and K63-linked ubiquitin. A, 293T cells were cotransfected with vectors encoding Flag-tagged WT Arkadia or C937A mutant along with Xpress-tagged ubiqutitin. Before lysis, cells were treated with MG132 for 4 h. Whole cell extracts were subjected to precipitation with Ni-NTA-agarose beads, followed by immuoblotting with anti-Xpress and FLAG antibody. B, in vitro ubiquitination assay. Flag-tagged WT Arkadia and C937A mutant were transfected into HEK293 cells and pulled down with anti-Flag antibody. Bead-bound proteins were subjected to in vitro ubiquitination assay in the presence of HA-tagged ubiquitin, E1, E2, and ATP, followed by Western blotting analysis. C, Arkadia ubiquitination reactions with WT, K6R, K11R, K27R, K29R, K33R, K48R, K63R, K0, or G76V ubiquitin were precipitated with Ni-NTA-agarose beads and analyzed by immunoblotting. Ub: ubiquitin. WCE: whole cell extract.