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Fig. 5. LBR binds to importin β via amino acids 45 to 90. (A) Schematic diagram of N-terminal deletion constructs of xLBR^1-210. The cDNAs for the different truncated N-terminal domains of xLBR were subcloned into pET28a to express the devised polypeptides. (B) Western analysis of the in vitro binding assay using the truncated LBR fragments and GST-importin β. Note that deletion past aa 45 reduced the binding, and past aa 90 abolished binding. (C) Quantification of the in vitro binding assayed in B by densitometry. 10% of the input for each fragment was set to 100%. The data is shown as the mean percentage bound plus the standard deviation. (D) Schematic diagram of the C-terminal deletion mutants of xLBR^1-210. The cDNAs for the different domains of xLBR were subcloned into pET28a-GFP to express the polypeptides fused with GFP. (E) In vitro binding assay using the C-terminally truncated LBR proteins and importin β. Note that GFP-xLBR^1-53 did not bind to importin β and GFP-xLBR^1-81 had weak binding. (F) Quantification of the in vitro binding assayed in E, which was identical to the method used in C. (G) Analysis of the binding of GFP-xLBR^45-90 to importin β. GFP-xLBR^45-90 bound to importin β as efficiently as GFP-xLBR^1-210. (H) 1. GFP-xLBR^45-90 pulled down importin β from mitotic HeLa extract. 2. GFP-xLBR^45-90 pulled down importin β from Xenopus egg extract.

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