Figure 2. Structure and nanomechanical properties of NPCs.

a, Confocal microscopy of isolated Xenopus laevis nuclei washed in buffer without (Control) or with Ran-/E-mix. Rch1-IBB-MBP-GFP (“IBB-GFP”) was added to the nuclei before treatment, and accumulated in the nuclei upon addition of Ran-/E-mix, while 70 kDa dextran remained excluded, indicating that the nuclei were intact. b, Confocal microscopy images of nuclear envelopes isolated from nuclei washed in buffer without (Control) or with Ran-/E-mix, and (the nuclear envelopes) subsequently exposed to Benzonase (“Benz.”). Nucleic acids were labelled with SYBR Gold, and fluorescent wheat germ agglutinin (WGA) was used to label NPCs. Scale bars in a, b: 400 μm. c, Quantification of IBB-GFP fluorescent signal in the nuclei (n = 15 for control and n = 16 for Ran-/E-mix data, with standard errors of the mean). d, Quantification of the SYBR Gold fluorescent signal at the nuclear envelopes, normalized to the WGA signal (n = 11 for control and n = 10 for Ran-/E-mix+Benz. data, with standard errors of the mean) for samples treated as described in a, b. e, Western blot from isolated nuclear envelopes showing a reduction of RNP K/J and IBB-GFP following Ran-/E-mix and Benzonase treatment; Nup153 was used as a loading control. f, Averaged stiffness cross sections of NPCs following washes without (Control) and with Ran-/E-mix and Benzonase. Black, dashed lines indicate the averaged profiles of the un-indented NPC surface. The same data are also shown as stiffness (in pN/nm) versus vertical position (z), for different radial positions, offset for clarity. g, Averaged stiffness cross sections of NPCs measured before (Control) and after 10 minutes incubation of the isolated nuclear envelopes with 0.5 μM importin β (Impβ). Number of NPCs in each data set: n = 33 (f, Control); n = 36 (f, Ran-/E-mix+Benz.); n = 35 (g, Control); n = 40 (g, Impβ). Colour scale: 0-10 pN/nm (f); 0-16 pN/nm (g).