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. 2021 Jan 6;20:100019. doi: 10.1074/mcp.RA120.002414

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© 2021 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Fig. 2 — In vitro ubiquitylation assays showing AriH2-induced ubiquitylation.A, Coomassie Blue–stained gel of various ubiquitylation reactions. All reactions contained MgATP, UBE1, and Ub. Lanes 1, 3, 5, 7, 9 contain CKB-ASB9-ELOB/C-CUL5-RBX2. Lanes 2, 4, 6, 8, 10 contain CKB-ASB9-ELOB/C-CUL5(NEDD8)-RBX2. Lane-specific additional components are UBE2D2 (lane 1, 2), UBE2R1 (lanes 3, 4), ARIH2-UBE2L3 (lanes 5, 6), ARIH2-UBE2L3-UBE2D2 (lanes 7, 8), and ARIH2-UBE2L3-UBE2R1 (lanes 9, 10). B, anti-CKB blot for the reactions as in panel A showing the increase in CKB migrating at high molecular weight. C, image J analysis of the Anti-CKB blot in B. The high-molecular-weight CKB ratioed against CKB reveals that the most efficient combination of components for polyubiquitylation of CKB is ARIH2 with UBE2R1. Subsets of this experiment (biological replicates) were repeated three times.