Characterization of the binding of CRM1 or importin β to Tpr or C-Tpr. Panel A: Binding was analyzed in the absence of RanGTP and NES (empty circles) or presence of RanGTP and NES (filled triangles) or absence of RanGTP and presence of NES (filled circles). Increasing eqimolar concentrations of CRM1, RanGTP and NES were incubated with Tpr and bound CRM1 was quantified as described in materials and methods. Panel B: Binding to Tpr was analyzed with increasing concentrations of importin β (filled circles) or importin β I178D (inverted triangles). Binding to C-Tpr was analyzed with increasing concentrations of importin β. The results are from duplicates of a single typical experiment. The standard deviation was <5% of the mean. The data were fitted to the equation B(Y) = Bmax × Y/(Kd + Y) (using Kaleida Graph software) where Y is the concentration of CRM1 (Panel A) or importin β/importin β I178D (Panel B) and B is the amount of CRM1(Panel A) or importin β/importin β I178D (Panel B) specifically bound. The correlation coefficients of the data to the fitted curves were >0.99.