Monitoring APC/C-Dependent Ubiquitylation

Qualitative assays to monitor APC/C-dependent ubiquitylation were performed as previously described [91 (link)]. In brief, reactions were mixed on ice, equilibrated to room temperature before the reactions are initiated with Ub or meUb, and quenched at the indicated time points with SDS. TTF2 ubiquitylation was monitored by mixing 100 nM APC/C, 1 μM Cdh1, 5 μM UBE2C, 5 μM UBE2S (when indicated), 1 μM UBA1, 5 μM TTF2, 5 mM Mg-ATP, and 150 μM Ub or meUb (S2 Fig). Ubiquitylation of UHRF1 wild type or its variants by APC/C were performed with 100 nM APC/C or pE-APC/C, 1 μM Cdh1 or Cdc20, 0.4 μM UBE2C, 0.4 μM UBE2S (when indicated), 1 μM UBA1, 0.4 μM UHRF1, 5 mM Mg-ATP, and Ub or meUb (Fig 4 and S4 Fig). Following SDS-PAGE, ubiquitylation products of the fluorescently labeled substrates were resolved by SDS-PAGE and imaged with the Amersham Typhoon 5 (Cytiva Life Sciences, Logan, Utah).