Kap enrichment in vivo and removal by Ran mix. (A) Transient transfections of MDCK cells with Kapβ1-EGFP, EGFP-CRM1, and Imp5-mCherry constructs reveal the subcellular localization of Kaps in vivo. Kapβ1 and CRM1 show visible nuclear rim stains indicating their enrichment at the NPCs, whereas Imp5 does not. (B) Fluorescence profiles obtained along the dashed lines shown in A. Kapβ1-EGFP and EGFP-CRM1 show fluorescence spikes (black) that coincide with the edges of the nuclear DAPI staining (blue), whereas similar features are lacking in the Imp5-mCherry signal. Line plots were created using Fiji after smoothing the images with a median filter (2-pixel radius) to minimize noise. (C) Retention of Kapβ1-EGFP, EGFP-CRM1, and Imp5-mCherry at NPCs following digitonin permeabilization and Ran mix treatments. Ran mix–treated cells are shown with original and brightness-adjusted settings for improved visualization. Each series of images was collected using the same imaging conditions. (D) Digitonin and Ran mix treatments significantly reduce the enriched pool of Kapβ1-EGFP (n = 10) and EGFP-CRM1 (n = 10) at the NE. Data points were normalized to the predigitonin NE fluorescence values of each cell. Note: Retention of Imp5-mCherry in digitonin-permeabilized HeLa cells lies below the detection limit, as shown with the brightness adjusted in C. The brightfield image confirms that cells were not removed from the field of view. Further quantification of Imp5-mCherry has been omitted. Error bars denote minimum and maximum measured values. Scale bars, 10 µm.