Kapβ1, CRM1, and Imp5 bind to NPCs in a concentration-dependent manner. (A) Experimental sequence. (B–D) Representative images of permeabilized HeLa cells incubated in increasing concentrations of (B) exoKapβ1, (C) exoCRM1, and (D) exoImp5. The concentration-dependent accumulation of each Kap is measured from their respective nuclear rim stainings. Cells in the first row are shown with the same dynamic range settings. The brightness is adjusted in the second row to improve visualization of the nuclear rim. Percentages indicate the laser power used to image the cells. Fluorescent beads were used for signal normalization to facilitate comparisons between images (see Materials and methods). Representative images were chosen from the same dataset. (E) Quantification of exoKapβ1 (green), exoCRM1 (blue), and exoImp5 (magenta) at the NPCs and normalized by the maximum fluorescence measured for each Kap at 10 μM. The apparent binding affinity of each Kap to the NPCs was obtained by fitting a single-component Langmuir isotherm to each respective dataset. Data points, error bars, and K_D,NPC values were obtained by propagating means and errors across all replicates (n ≥ 3). Scale bars, 20 µm.