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. 2018 Aug 17;293(39):15304–15315. doi: 10.1074/jbc.RA118.004444

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© 2018 Xu et al.

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Figure 2. — K3xR variant shows auto-ubiquitination activity. A–C, His-tagged K3xR protein (NopM with K502R, K531R, and K537R substitutions) was used for ubiquitination reactions (2 h), and auto-ubiquitination was detected with the anti-NopM antibody. Nonubiquitinated K3xR is marked by arrowheads. A, reactions (2 h) were performed with (+) or without (−) ATP. B, incubation of reaction products with and without thrombin (removal of the N-terminal His tag). C, reactions with GST-tagged ubiquitin, His-tagged ubiquitin, with a mixture (Ub mix; GST- and His-tagged ubiquitin, 1:1), or with UbΔGG (Ub without C-terminal di-glycine residues). D, analysis of intermolecular transfer of His-tagged ubiquitin from GST–NopM to His-tagged K3xR-C338A (NopM with C338A, K502R, K531R, and K537R substitutions). Nonubiquitinated His–K3xR–C338A is marked by an arrowhead, mono-ubiquitinated His–K3xR–C338A by an asterisk, and GST–NopM by an arrow. WB, Western blot.