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In vitro ubiquitination assay The ubiquitination assays were performed in 30 l reaction volume containing 0.45 M E1 (Uba1), 45 M ubiquitin, 5 mM ATP, 4.5 M Ube2d3 or Ube2e2 (WT, C139S or C139A), 1 M CULLIN4-RBX1-DDB1 or neddylated-CULLIN4-RBX1-DDB1 complex, and 1 M COP1-c-Jun or DET1-COP1-c-Jun or DDD-Ube2e2-COP1-c-Jun complex

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