A, B, C, Titration of different types of diUb with GB1-AT3-UIM12. A, Linear diUb; B, K48-linked diUb; C, K63-linked diUb. By fitting the data with the sequential binding model, these titrations are well-fitted to give two distinct binding affinities. D, E, F, Titration of K48-linked diUb with different AT3-UIM12 forms. D, GS-Sub; E, GS-Ins; F, RAP80-Linker. The concentration of K48-linked diUb is 100 µM and those of the AT3-UIM12 stocks are 2 mM. The titrations are well-fitted to a usual two-site binding model (dashed line) rather than the sequential binding model (solid line). The concentrations of diUb types are 100 µM and those of AT3-UIM12 stocks are 2 mM. The GB1 fusion was used for peptide expression and quantification.