Ubiquitin Chain Cleavage Assay

Ub chain cleavage assays were performed as previously described (Elliott et al., 2016 (link)) using purified Met1- and Lys63 -linked tetra ubiquitin chains. CYLD variants were prepared at twice the desired enzyme concentration. In the case of IKKβ activation, CYLD pre-treated with lambda protein phosphatase that was subsequently purified by size exclusion chromatography resulting in purified de-phosphorylated CYLD, was incubated with 10 mM Mg-ATP with or without IKKβ for 30 min prior to the DUB assay. Reactions were performed at RT and the reaction was initiated through the addition of DUB to ubiquitin substrate. At designated time points, samples were taken and the reaction was quenched through addition of SDS sample buffer and analyzed by SDS-PAGE and stained using silver stain kit (Biorad).