Figure 1. Ubiquitin chain interactor enrichment using different deubiquitinase inhibitors.

(A) Schematic of the ubiquitin (Ub) interactor enrichment method. In short, Ub chains are enzymatically synthesized, purified, and conjugated to biotin. Ub chains are immobilized on streptavidin resin and incubated with chloroacetamide- or N-ethylmaleimide–treated cell lysate in quadruplicate. Pulldown with bead only is used as a negative control for background binders. Interactors are enriched and unspecific binders washed off. Enriched interactors are identified by LC-MS/MS and analysed using MaxQuant and R, including a prefiltering step to select for proteins enriched on Ub over the bead-only control. (B) Western blot of Ub interactor pulldown using chloroacetamide or N-ethylmaleimide, as deubiquitinase inhibitors. Silver stain of input Ub. Pulldown blotted using anti-RAD23B, anti-EPN2, and anti-Ub antibodies. Input lys is input lysate. Ctrl is bead-only control pulldown.