RanGTPase shuttling between the nucleus and cytoplasm is enhanced by conditions that increase available GTP. (A) Representative confocal images of GFP-Ran cells depleted of SUN2, where FLIP was used to bleach the cytoplasm and measure the loss of fluorescence intensity from the nucleus. (B–I) Graphs of fluorescence change during FLIP (B, D, F, and H) and rate of loss in fluorescence of GFP-Ran over the first 5 min (C, E, G, and I) for cells depleted of SUN2 ([B and C] siControl n = 51, siSUN2 n = 58; three independent replicates), (D and E) treated with cytochalasin B (control n = 64, cytoB n = 60; three independent replicates), (F and G) plated for 2 or 24 h (2 h n = 23, 24 h n = 28; three independent replicates), or (H and I) treated with cycloheximide (control n = 38, cycloheximide n = 26; two independent replicates). Significance was calculated using unpaired t test (C, E, G, and I). P***<0.001, P****<0.0001. Scale bar 10 µm. Error bars represent ± SEM.