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. 2013 Apr 5;288(21):15181–15193. doi: 10.1074/jbc.M112.446534

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

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FIGURE 2. — Amot130 is ubiquitinated by AIP4. A, the relative levels of ubiquitinated Amot130 and AIP4 following immunoprecipitation of HA-tagged Lys-0 ubiquitin (HA-K0 Ub) in HEK 293T cells co-expressing with and without YFP-tagged Amot130, Myc-tagged AIP4, or control vectors were detected by immunoblot. B, endogenous Amot was immunoprecipitated from HEK 293T cells expressing HA-tagged ubiquitin (HA-Ub) and Myc-tagged AIP4 or the catalytically inactive AIP4 (C830A). The levels of total and ubiquitinated Amot were detected by immunoblot (IB). The ratio of pixel intensities of ubiquitinated Amot130 over total Amot130 is indicated. C, the relative incorporation of HA-ubiquitin into immunoprecipitated Myc-tagged AIP4 or mutant AIP4 (C830A) in HEK 293T cells following co-expression with FLAG-tagged Amot130 was detected by immunoblot. D, HEK 293T cells expressing HA-ubiquitin and Myc-tagged AIP4 or mutant AIP4 (C830A) as well as YFP-tagged Amot130 or control vector were lysed in phospholipase C buffer and soluble HA-ubiquitin-modified proteins were detected by immunoblot. Molecular mass standards (in kDa) are indicated.