Kapβ1 depletion softens the NPC permeability barrier to nonspecific cargoes in vivo. (A) Representative image sequence showing the recovery of 2xEGFP in the nucleus obtained during a FRAP experiment in control siRNA-treated cells. Lightning indicates the nuclear photobleaching event at t = 0. Scale bar, 10 µm. (B) Fluorescence recovery curves (symbols) and their fits (black lines) as obtained in individual cells. In all cases, an increase in nuclear fluorescence (normalized fluorescence <1) correlates to a concomitant decrease in cytoplasmic fluorescence (normalized fluorescence >1). Both nuclear recovery and cytoplasmic loss of fluorescence are characterized by similar time constants because Kaps do not play a role in the passive diffusion of 2xEGFP. For clarity, only every 10th data point is shown. (C) Kapβ1 silencing expedites the passive exchange of 2xEGFP cargoes across NPCs. (D) Kapβ1 silencing leads to an increase in NPC permeability for 2xEGFP cargoes. Statistical analysis was performed using ordinary one-way ANOVA. P adjusted values were calculated using the Benjamini-Hochberg procedure (***, P = 0.0002; **, P = 0.0021; *, P = 0.0332; ns = 0.1). See main text for details.