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. 1997 Mar 4;94(5):1745–1748. doi: 10.1073/pnas.94.5.1745

Figure 2.

Figure 2

Effect of cytohesin-1 on [35S]GTP[γS] binding to ARF3. Native ARF3 (0.25 μg, ≈12.5 pmol) was incubated with zero (□), 0.06 (•), 0.12 (▴), or 0.24 μg (▾) of cytohesin-1 and 4 μM [35S]GTP[γS] (2 × 106 cpm) in 100 μl containing 20 mM Tris (pH 8.0), 10 mM imidazole, 100 mM NaCl, 10 μg of phosphatidylserine, 40 μg of BSA, 10 mM DTT, and 3.1 mM MgCl2 at 36.5°C for the time indicated, then at 0°C for a total of 60 min. Proteins were collected on nitrocellulose filters for radioassay of bound GTP[γS]. GTP[γS] bound to samples without ARF3 or cytohesin-1 after 60 min at 0°C was 0.1 pmol, to ARF3 was 0.14 pmol, and to cytohesin-1 alone was 0.11 pmol. After 60 min at 0°C, 12.5 pmol of ARF3 plus 0.06 μg, 0.12 μg, or 0.24 μg of cytohesin-1 bound 0.99, 1.2, and 1.53 pmol, respectively. Control values have been subtracted from the data presented.