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Schematic of the fluorescence polarization-based competition (FPcomp) assay. (A) The binding affinity of Ub-fluorescein and UCH37C88A•RPN13 was measured through a direct binding assay. (B) The binding affinity of unlabeled mono-Ub, Lys48 linked Ub2, Ub3, and Ub4 was then measured by a competitive binding assay, in which the unlabeled Ub derivative competes for binding with the fluorescein-labeled Ub.
