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. 2001 Mar;12(3):539–549. doi: 10.1091/mbc.12.3.539

Figure 2.

Figure 2

Association of importin β-like proteins with Gsp1p-GTP and Yrb1p. (A) GST-Gsp1p-GTP (5 μg per reaction) was immobilized on glutathione Sepharose and incubated for 30 min at 4°C with 3 μg of Yrb1p or 12 μg of Xpo1p, Cse1p, Kap95p, Pse1p, Yrb4p, or Kap104p, as indicated. The reactions containing the export receptors Xpo1p and Cse1p received additionally 4 μg of PKI-NES-2GFP (NESp) or 5 μg of importin α (Srp1p). Molecular weight markers (M) are in kDa. (B and C) GST-Yrb1p (4 μg per reaction) was immobilized on glutathione Sepharose and incubated for 30 min at 4°C with 12 μg of each importin β-like protein and with 10 μg of Gsp1pQ71L loaded with either GTP (B) or GDP (C). The load of Gsp1p (50% of the input) is shown in the left lane. Asterisks indicate weak binding of Kap104p and Kap95p to Yrb1p in the presence of Gsp1p-GDP. The bound material of all reactions was washed three times and analyzed by SDS PAGE and Coomassie blue staining.