Fig. 9: Pull-down assay of importin β and FG-nups.
From: Ran modulates allosteric crosstalk between importin β surfaces
Each panel shows (top) representative cropped Coomassie-stained SDS-PAGE gels from pulldown assays and (bottom) the corresponding quantification obtained by densitometric analysis of bands from four independent experiments (n = 4). A GST-Nup358 (res. 2503–2893), B GST-Nup62, and C GST-Nup153 (res. 946–1472) were coupled to glutathione agarose beads and incubated with equal amounts of purified importin β alone (lane 3), or importin β with αIBB (lane 4), Ran-GTP (lane 5), or Ran-GTP plus RanBP1 (lane 6). The intensity of importin β bands bound to GST-FG-nups was quantified using ImageJ78 across four independent SDS-PAGE experiments (n = 4). Histogram columns represent the ratio of importin β band intensity (black circles) normalized by the intensity of the GST-FG-nup band, with relative standard deviation indicated by error bars. Histograms are presented as mean values +/− standard deviations. A Shows that 45.6 ± 6.9% of importin β was pulled down by GST-Nup358 beads. This value dropped to 36.0 ± 6.6% in the presence of MBP-αIBB, 14.8 ± 4.9% with Ran-GTP, and 7.9 ± 4.2% with Ran-GTP:RanBP1. B Shows that 70.9 ± 11.4% of importin β was pulled down by GST-Nup62, decreasing to 40.4 ± 12.3% with MBP-αIBB, 5.3 ± 0.9% with Ran-GTP, and 4.0 ± 0.9% with Ran-GTP:RanBP1. C Shows that 83.1 ± 11.2% of importin β was pulled down by GST-Nup153 beads, and slightly more, 88.5 ± 13.6%, in the presence of MBP-αIBB. Importin β binding to Nup153 dropped to 23.7 ± 6.1% with Ran-GTP and 17.6 ± 5.1% with Ran-GTP:RanBP1. D–F Show comparable pulldown experiments as in A–C but using Ran-GDP. Specifically, D GST-Nup358 (res. 2503–2893), E GST-Nup62, and F GST-Nup153 (res. 946–1472) coupled to glutathione agarose beads were incubated with equal amounts of purified importin β (lane 3) alone, or importin β together with αIBB (lane 4), Ran-GDP (lane 5) or Ran-GDP plus RanBP1 (lane 6). D Shows that 62.9 ± 8.4% of importin β was pulled down by GST-Nup358 beads; 56.8 ± 4.6% in the presence of MBP-IBB, 55.2 ± 5.6% with Ran-GDP, and 57.2 ± 19.6% with Ran-GDP:RanBP1. E Shows that 107.0 ± 16.7% of importin β was pulled down by GST-Nup62, 78.2 ± 8.2% with MBP-αIBB, 104.7 ± 17.7% with Ran-GDP, and 56.9 ± 11.4% with Ran-GDP + RanBP1. F shows that 88.4 ± 10.8% of importin β was pulled down by GST-Nup153 beads; 83.8 ± 25.3% with MBP-αIBB, 78.1 ± 10.2% with Ran-GDP, and 55.8 ± 14.6% with Ran-GDP + RanBP1. For all pulldown gels A–F, only fractions bound to each FG-nup and recovered on beads are shown. M.W. molecular weight markers. See Supplementary Fig. 8 for the uncropped gels. A significant (p < 0.01) deviation from the importin β:GST-FG-nup ratio was assessed using a two-tailed Student’s t test, assuming unequal variance. In this analysis, *p < 0.05 and ***p < 0.001 are considered statistically significant, while n.s. indicates not significant. Source data, including all SDS-PAGE gels, are provided as a Source data file.
