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. 2002 Apr 16;99(8):5436–5441. doi: 10.1073/pnas.062059999

Figure 4.

Figure 4

Bfa1 or Bub2 directly interacts with Tem1 in vitro. To investigate interactions among recombinant Bfa1, Bub2, and Tem1 proteins under various conditions, partially purified 0.5 μg each of T7-Bfa1 and T7-Bub2 proteins was added into reaction tubes containing either bead-bound GST or GST-Tem1, which is supplemented with GDP, GTP[γS], or GDP + AlFInline graphic. Proteins associating with either GST-Tem1 or GST were precipitated and detected by immunoblotting with an anti-T7 antibody. The same membrane subsequently was blotted with an anti-GST antibody to detect the precipitated GST and GST-Tem1 fusion protein. Control GST blot was assembled with GST-Tem1 blot from the same exposure, and the migration difference does not reflect the actual mobility differences of these two proteins in the gel. Input, 15% of the proteins used in the binding assays; control, GST control with both Bfa1 and Bub2 in the presence of GDP+AlFInline graphic. GDP and GTP[γS] were supplemented at the final concentration of 100 μM, whereas AlFInline graphic was supplemented at 110 μM. Bfa1, T7-Bfa1-His-6 recombinant protein; Bub2, T7-Bub2-His-6 recombinant protein.