Figure 2.

Interaction of wild type and E1-Ran with RanBP1 and karyopherin β, assayed by ligand blotting. (A) Gel transfer assay. A 0.5-μg sample of GST-RanBP1 and 1-μg samples of GST, karyopherin β, and a truncated karyopherin β lacking its amino terminal 12 residues (Δ karyopherin β) were resolved by SDS-PAGE, transferred to nitrocellulose, renatured in situ, and probed with wild-type Ran·[α³²P]GTP alone, wild-type Ran·[α-³²P]GTP + RanBP1, or E1-Ran·[α-³²P]GTP alone. Filters were subjected to autoradiography for 3 h. Mobilities of size markers in kDa are indicated on the left. The duplicate Coomassie blue-stained gel shows the relative intactness of the karyopherin β preparations. (B) Dot blot assay. One- or 0.4-μg samples of nondenatured GST-RanBP1, GST, karyopherin β, and Δ karyopherin β were spotted directly onto nitrocellulose and probed with wild type and E1-Ran charged with [α-³²P]GTP in the presence or absence of added RanBP1 as indicated on the left. Filters were subjected to autoradiography for 4 h. The 1-μg and 0.4-μg labels on the right indicate the protein content of the adjacent eight dots.