Binding of CRM1 and Imp5 to FG Nup layers in vitro. (A and B) SPR response curves obtained for (A) CRM1 and (B) Imp5 binding to cNup153 (green), cNup62 (black), cNup98 (blue), and cNup214 (red). Vertical jumps in the signal correspond to triple BSA injections used to measure FG Nup layer height. RU, resonance units. (C) Equilibrium binding analysis of Kapβ1 (yellow), CRM1 (blue), and Imp5 (magenta) to cNup62, cNup98, cNup153, and cNup214. Lines represent single-component (solid) or two-component (dashed) Langmuir isotherm fits to the average SPR equilibrium response (R_eq). The mean apparent dissociation constant calculated from n ≥ 4 replicates was used for the fitting. For each replicate, data points were normalized to the maximum response value (R_max) or their sum (R_max1 and R_max2) obtained from the equilibrium fit. Note: The Kapβ1 data were reproduced from Kapinos et al. (2017).