Fig. 7. LBR-containing NE precursor vesicles are recruited to participate in NE assembly through importin β. (Aa). Importin β-coated Sepharose beads were blocked in advance with BSA or xLBR^1-210 and used in the NE assembly assay in mitotic HeLa extract prepared from constitutive GFP-xLBR-expressing HeLa cells. Note that the incorporation of GFP-xLBR into NE could be blocked by xLBR^1-210 (indicated by arrow) but not by BSA (indicated by arrowhead). (Ab 1 and 2) Importin β-coated Dyna beads were blocked in advance with BSA (Ab 1) or xLBR^1-210 (Ab 2) and used in the NE assembly assay, followed by analysis with TEM. The double-layered NE (arrows) could be clearly seen. Note that xLBR^1-210 blocked the NE precursor vesicle recruitment and the NE assembly around the importin β-coated beads (Ab 2). Scale bar, 500 nm. (Ab 3) Negative staining of the NE with typical NPCs assembled on the surface of importin β-coated TEM grids. NPCs are indicated by asterisks. (Ba) RanGDP-coated beads induced NE assembly in mitotic HeLa extract prepared from constitutive GFP-xLBR-expressing HeLa cells in the presence of His-xLBR^90-210 or His-xLBR^1-210. The result showed that His-xLBR^1-210 (arrow) but not His-xLBR^90-210 (arrowhead) could specifically prevent recruitment of GFP-xLBR-bound NE precursor vesicles onto the RanGDP-beads to assemble the NE. (Bb) Statistical analysis of the NE assembly in the presence of His-xLBR^90-210 or His-xLBR^1-210. The data is shown as the mean percentage decorated beads plus the standard deviation. (C) NE assembly assay in the extract prepared from constitutive GFP-xLBR-expressing HeLa cells and depleted of importin β with Ran^Q69L. (Ca) Western blot analysis showed that more than 90% of the endogenous importin β in the extract was depleted by Ran^Q69L-GTP. The untreated extract (1), extract mock-depleted with control beads (2), extract depleted with Q69L beads (3), proteins bound to control (4) or Q69L (5) beads were subjected to Western blot analysis for importin β. (Cb) When NE assembly was induced with RanGDP-beads in the importin β-depleted HeLa extract, the NE assembly was efficiently blocked (indicated by arrow) compared with that in the mock-depleted extract (indicated by arrowhead). If importin β-coated beads were added to the importin β-depleted extract, the NE assembly could occur efficiently (indicated by arrowhead,). The addition of herparin to a final concentration of 0.5% did not influence the NE assembly around importin β-coated beads. (Da) When His-xLBR^90-210 or His-xLBR^1-210 was added to the importin β-depleted extract, the NE assembly around the added importin β-beads could only occur in the extract containing added His-xLBR^90-210 (indicated by arrow) but not His-xLBR^1-210 (indicated by arrowhead), indicating His-xLBR^1-210 prevented the access of the NE precursor vesicles to the importin β on the beads. (Db) Statistical analysis of the NE assembly in the presence of His-xLBR^90-210 or His-xLBR^1-210. The data is shown as the mean percentage (plus the standard deviation) of the beads decorated with the NE.