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. 2020 Nov 18;10(11):200237. doi: 10.1098/rsob.200237

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© 2020 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 2. — S2-MacroD reverses PARP14-derived ADP-ribosylation. (a) PARP14 can efficiently modify itself in vitro. Auto- and trans-ADP-ribosylation activity (PARP14 WWE-CAT and PARP14 macrodomains 1–3 [MOD1-3], respectively) of PARP14 were assessed by incubation with ³²P-NAD⁺in vitro. Samples were analysed by SDS-PAGE followed by CBB staining and autoradiography (³²P) which reveal efficient automodification of PARP14 WWE-CAT as well as trans-modification of MOD2 and MOD3. (b) S2-MacroD can reverse PARP14-derived ADP-ribosylation. PARP14 WWE-CAT, MOD2 and MOD3 were modified as in (a) followed by incubation with S2-MacroD. Samples were analysed by CBB staining and autoradiography and show that S2-MacroD wild-type (wt) can efficiently remove ADP-ribosylation from all three proteins. (c) Pairwise sequence identity comparison of coronaviral MacroD domains with experimentally proven (ADP-ribosyl) hydrolase activity. Sequence identity and similarity (in parentheses) are provided.