Fig 1: Nup153 regulates NPC sensitivity to the mechanical directionality of the translocating protein cargo.a, Schematics of the NPC, showing the approximate location of Nup153, Nup98 and Nup214. b, Schematics of the dot blot assay. c, Dot blot membranes to assess protein binding to Nup153, Nup98 and Nup214, including a control (no Nup present). d, Dot blot intensity for each of the Nups interacting with Ig27WT, normalized by the control experiment. Each data point corresponds to the average intensity of three dots per membrane (N = 5 independent dot blot experiments). Significance levels for two-tailed t-test: NS, P > 0.05; **P < 0.01; Nup153 versus Nup214, P = 6.94 × 10−3; Nup214 versus Nup98, P = 0.76; Nup153 versus Nup98, P = 1.42 × 10−3. Boxes span from the first to the third quartile, and the horizontal line indicates the median. e, Dot blot intensity for Nup153 binding to either Ig27WT or (GS)4-Ig27WT. Significance levels fo the two-tailed paired t-test: *P < 0.05. WT versus (GS)4-WT, P = 0.04. f, Representative confocal images of U2OS cells 30 min into the recovery phase. Scale bar, 10 µm. g, Time courses of the relative nucleus-to-cytoplasm localization of R16-WT versus WT-R16 (top) and R16-Y9P versus Y9P-R16 (bottom) under normal conditions (siNT) and Nup153 knockdown (siNup153). h, Associated import rates: R16-WT (siNT) (kI = 1.35 ± 0.05 ks−1, n = 130), R16-WT (siNup153) (kI = 1.15 ± 0.05 ks−1, n = 108); WT-R16 (siNT) (kI = 1.20 ± 0.05 ks−1, n = 179), WT-R16 (siNup153) (kI = 1.09 ± 0.05 ks−1, n = 98); R16-Y9P (siNT) (kI = 1.51 ± 0.06 ks−1, n = 92), R16-Y9P (siNup153) (kI = 0.93 ± 0.06 ks−1, n = 57); Y9P-R16 (siNT) (kI = 1.07 ± 0.04 ks−1, n = 100), Y9P-R16 (siNup153) (kI = 0.83 ± 0.09 ks−1, n = 33). Significance levels for the two-tailed Mann–Whitney non-parametric test: NS, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001; siNT R16-WT versus siNup153 R16-WT, P = 2.14 × 10−3; siNT WT-R16 versus siNup153 WT-R16, P = 0.02; siNT R16-WT versus siNT WT-R16, P = 0.03; siNup153 R16-WT versus siNUP153 WT-R16, P = 0.25. siNT R16-Y9P versus siNup153 R16-Y9P, P = 5.70 × 10−9; siNT Y9P-R16 versus siNup153 Y9P-R16, P = 2.50 × 10−3; siNT R16-Y9P versus siNT Y9P-R16, P = 1.26 × 10−7; siNup153 R16-Y9P versus siNUP153 Y9P-R16, P = 0.17. i, Representative confocal image of U2OS cells. Scale bar, 10 µm. j, Time courses of the relative nucleus-to-cytoplasm localization of WT and (GS)4-WT under normal conditions (siNT) and Nup153 knockdown (siNup153). k, Associated import rates; WT (siNT) (kI = 1.61 ± 0.05 ks−1, n = 147), WT (siNup153) (kI = 1.22 ± 0.05 ks−1, n = 118); (GS)4-WT (siNT) (kI = 2.02 ± 0.05 ks−1, n = 169), (GS)4-WT (siNup153) (kI = 1.28 ± 0.04 ks−1, n = 122). Significance levels for the two-tailed Mann–Whitney non-parametric test: NS, P > 0.05; ****P < 0.0001; siNT WT versus siNup153 WT, P = 7.74 × 10−7; siNT (GS)4-WT versus siNUP153 (GS)4-WT, P = 2.50 × 10−28; siNT WT versus siNT (GS)4-WT, P = 1.42 × 10−6; siNup153 WT versus siNup153 (GS)4-WT, P = 0.59. l, Directionality calculated as the ratio between the average import rate (top) and accumulation (bottom) for translocating R16-Ig27Y9P versus Ig27Y9P-R16 constructs, measured when silencing individual FG-Nups. Error bars indicate s.e.m. m, Schematic of the proposed role of Nup153 in identifying locally unstructured protein regions. Significance levels for two-tailed t-test (d), two-tailed paired t-test (e), Mann–Whitney non-parametric test (h,k): NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All points and bar plots indicate mean ± s.e.m.