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. 1998 Dec 28;143(7):1813–1830. doi: 10.1083/jcb.143.7.1813

Figure 6.

Figure 6

Ran-GTP induces the release of Kap121p from Nup53p. (A) A Kap121-pA/ GST-Nup53p complex was formed in vitro by incubating yeast cytosol containing Kap121-pA with GST-Nup53p immobilized on GT– Sepharose beads (see Fig. 5 A). This complex was then incubated for 30 min at room temperature with buffer alone or Ran preloaded with GTP, GDP, or GTP-γ-S. Kap121-pA that was released from the column and that which remained bound were separated by SDS-PAGE and detected by Western blotting using donkey anti–rabbit conjugated to HRP and ECL. Shown are the relevant regions of the resulting autoradiogram. (B) The ability of Ran-GTP to release Kap121p from the isolated Nup53-pA complex (see Fig. 3) was examined. Equal amounts of the complex were incubated with Ran-GTP or Ran-GDP. Kap121p remaining bound to the complex and in the released fractions was detected by silver staining (Silver) and Western blot analysis using anti-Kap121p antibodies (α-Kap121). Relevant regions of the gel and autoradiogram are shown.