Fig. 6. LBR binding to importin β is regulated by the nucleotide state of Ran and is independent of importin . (A) An in vitro assay of for the release of GST-importin β-bound His-xLBR^1-210 in the absence (no Ran, lanes 1 and 2) and presence of His-Ran^Q69L-GTP (RanQ69L, lanes 3 and 4) or His-Ran^T24N-GDP (RanT24N, lanes 5 and 6). The soluble (S) and pelleted (P) (bound to GST-importin β) proteins were analyzed for His-xLBR^1-210 with anti-His antibody. (B) An in vitro assay for the release of GST-importin β^45-876-bound His-xLBR^1-210 in the absence (no Ran, lanes 1 and 2) and presence of His-Ran^Q69L-GTP (RanQ69L, lanes 3 and 4) or His-Ran^T24N-GDP (RanT24N, lanes 5 and 6) as in A. (C,D) Pull-down assays of the endogenous importin β of mitotic HeLa cell extract (C) or Xenopus egg extract (D) by CNBr-activated Sepharose 4B-coated with His-xLBR^1-210 in the presence of buffer alone, His-Ran^Q69L-GTP or His-Ran^T24N-GDP. The LBR-bound importin β was probed with the anti-importin β antibody on western blots. (E) An in vitro binding assay of purified GST-importin β with His-xLBR^1-210 in the absence or presence of importin . The LBR was probed with the anti-His antibody. (F) His-xLBR^1-210 binding assay with GST-importin β or GST-importin β^1-462 in vitro. His-xLBR^1-210 was probed with the anti-His antibody.