NXF1 is constantly present at the NPC. (A) Coimmunostaining of endogenous NXF1 and Nup214, imaged by confocal microscopy focusing on the middle plane of the nucleus. (B) Immunofluorescence of hnRNP A1, UAP56, and CBP80 in cells permeabilized after fixation (left) or before fixation (right; see Materials and methods). DIC, differential interference contrast. (C) Colocalization of NXF1 (magenta) with the NPCs (Nup358, green) in U2OS cells imaged by STED, focusing on the upper surface of the nucleus. Scale bars, 5 µm (left) and 0.5 µm (right). (D) FRAP experiment on U2OS cells stably expressing GFP-NXF1. Cells were imaged and photobleached in the nucleoplasm or at the NPCs. Fluorescence recovery was tracked over time and plotted. See Materials and methods for the number of cells shown. Fit of average of three repeated experiments is presented (script FRAP_fit in the online supplemental materials). (E) Photoactivatable PAGFP-NXF1 was expressed in cells. A nucleoplasmic region was activated, and the NXF1 signal (black) accumulating in the NPCs was tracked over time. Frames from Video 8. Scale bars, 5 µm.