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In vitro ubiquitylation assays and in vitro ubiquitylation/deubiquitylation competition assays were performed (37 C, 4 h or the indicated number of hours) with 5 g ubiquitin, 50 ng E1, and 50 ng UBCH7 or UbcH5c (UBE2D3) as indicated, along with the indicated amount of TRIP12, UBR5, or OTUD5, in a 20 L reaction [reaction buffer: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 2 mM ATP, and 0.5 mM DTT]

A comprehensive literature search was conducted using PubMed, Web of Science, and CNKI