The ciliary diffusion barrier is distinct from the barriers at the axon initial segment and the NPC. (A) Cells were treated with 4 µM Cytochalasin D for 30 min to depolymerize actin before digitonin permeabilization and incubation with Luc-GBP for 10 min. Arrowheads point to the base of the cilium in the GBP channel. (bottom) F-actin (phalloidin staining), with the gain increased in untreated cells relative to Cytochalasin D–treated cells for clarity. (B) Entry of GFP (shown in white) into nuclei of digitonin-permeabilized HeLa cells was assessed in the presence and absence of 5% wt/vol 1,2-trans-cyclohexanediol. (C) The rate of MBP-GBP entry into cilia of digitonin-permeabilized IMCD3 cells was assessed in the presence and absence of 5% wt/vol cyclohexanediol. Traces for individual cilia are shown as thin lines (n ≥ 10). Fitted exponential curves corresponding to the mean entry rates are shown as thick lines. (D) Effect of 4 µM dominant-negative Importin-β (residues 45–462) and 75 µg/ml WGA on entry of GFP into nuclei of digitonin-permeabilized HeLa cells (right) and on entry of MBP-GBP into cilia of digitonin-permeabilized IMCD3 cells (left). (E) Comparison of rates of MBP-GBP capture at the proximal segment of primary cilia for untreated cells and cells treated with WGA or 1,2-trans-cyclohexanediol. Error bars indicate standard deviations (n ≥ 10). Rel., relative. (F) Effect of anti-Nup antibody mAb414 on MBP-GBP entry into cilia of digitonin-permeabilized IMCD3 cells. The arrowhead points to the cilium base in the mAb414 channel, where no staining is seen. Bars: (main images) 5 µm; (insets) 1 µm. All insets show primary cilia with channels shifted to aid visualization.