Fig. 4. LBR binds to importin β in vivo through the N-terminal 1-210 domain. (A) Interphase and mitotic HeLa cells were analyzed by indirect immunofluorescence microscopy using anti-importin β and LBR antibodies, and shows colocalization of these endogenous substances. (B) The N-terminal domain of xLBR binds to importin β in mitotic HeLa extract. CNBr-activated Sepharose 4B-coated with GFP-xLBR^1-210 was incubated with mitotic HeLa extract. The proteins bound to the beads were isolated and subjected to western blot analysis for the presence of importin β. (C) A fluorescence protein binding assay shows direct interaction of LBR with importin β. Purified GFP-xLBR^1-210 bound to importin β but not GFP beads. (D) The purified N-terminal domain of xLBR binds to importin β in the presence of 100 mM or and 300 mM NaCl but not in the presence of 500 mM NaCl. Proteins associated with the importin β beads were subjected to western blot with anti-His antibody. (E) In Xenopus egg extract, GFP-xLBR^1-210 but not GFP beads could specifically pull-down the endogenous importin β (arrowhead). Proteins associated with the beads were separated on a SDS gel followed by silver staining. The molecular mass markers are shown on the left. (F) Western blot analysis of the precipitated protein in E and egg extract for the presence of importin β.