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. 2013 Oct 7;288(47):33722–33737. doi: 10.1074/jbc.M113.483305

FIGURE 5.

FIGURE 5.

The EDA-ID-associated D406V mutation impairs the binding for Lys-63- and linear di-ubiquitin. A, size exclusion chromatography analysis of WT and D406V mutant ZF interactions with various forms of di-Ub chains (linear (Met-1) and Lys-48- and Lys-63-linked di-Ub). The experimental conditions were as in Fig. 3C, including the void (V0) and internal (Vi) volumes of the column. B, binding curves of WT F-ZF (black circles) and its D406V mutant (red squares) with linear or Lys-48- or Lys-63-linked di-ubiquitin measured by fluorescence polarization at 25 °C. Binding constants (KD) extracted from non-linear least-squares fitting (solid line) are indicated for each di-ubiquitin taking into account a complex stoichiometry of 1:1. Error bars represent S.D. from three independent experiments performed with 0.1, 1, or 10 μm WT and mutant F-ZF.