FIGURE 3.
Deletion of the central repeat (7R) domain prevents intramolecular TACC3 complex formation. A, primary structure of TACC3-ΔR lacking the 7R domain. B, elution profile of TACC3-ΔR on analytical gel filtration (Superpose 6, 10/300) before (solid line) and after (dashed line) thrombin cleavage. Vo, void volume. C, peak elution fractions were analyzed on SDS-PAGE (4–15% gradient gel) followed by CBB staining (upper panel) and immunoblotting using the indicated antibodies (middle and lower panels). Thr., thrombin. D, the interaction between the isolated repeat region (7R) and the TACC domain (CC) was analyzed by pulldown assays and immunoblotting using N18 and C18 antibodies against TACC3. The asterisk indicates bovine albumin used to reduce unspecific binding to GSH-Sepharose beads. E, analysis of the interaction between 7R and subdomains of the TACC domain (CC1, CC2) employing ITC. Heat changes after association of the indicated protein fragments indicate that 7R selectively interacts with CC2 (upper panel) but not CC1 (lower panel). See supplemental Fig. S6 for experimental ITC controls.