Figure 6

The import Mig2-Kap95 complex formation in vitro. GST pull-down analysis of Mig2 (a) and Mig2nls1 (b) interaction with Kap60 and Kap95 purified proteins. (c) Effect of Gsp1 on the Mig2-Kap95 interaction stability. GST-Mig2, GST-Mig2nls1, GST-Gsp1, GST-Kap60 and GST-Kap95 fusion proteins were purified on glutathione-Sepharose columns and incubated with thrombin to release, respectively, Gsp1, Kap60 and Kap95 proteins. The Gsp1 protein was loaded with GTP to generate Gsp1(GTP) or with GDP to generate Gsp1(GDP). The purified proteins were incubated with purified GST-Mig2 or GST-Mig2nls1 bound to glutathione-Sepharose beads in the absence (a and b) or in the presence (c) of Gsp1(GTP) and Gsp1(GDP). The beads were washed extensively. Co-precipitated proteins were resolved by 12% SDS-PAGE and visualized on a Western blot with polyclonal anti-Kap60 or anti-Kap95 antibodies. The level of Kap95 protein present in the different extracts used in Figure 5c was determined by Western blot using anti-Kap95 antibodies. The Western blots shown are representative of results obtained from four independent experiments.