Figure 1
DDA3 is an EB1-binding protein.
(a) GST-EB1 on glutathione beads pulled down purified His-DDA3. Western blotting using anti-His antibody indicated a specific interaction. CB, Coomassie blue stain. (b) Co-immunoprecipitation of FLAG-DDA3 and EB1-GFP from human embryonic kidney 293T cells. Western blotting analysis with anti-GFP antibody indicated the specific association. (c) Schematic drawing of EB1 deletion mutants. Residue numbers at domain boundaries are indicated. CH, calponin homology domain; CC, coiled-coil region. (d) DDA3 interacted with the C-terminal domain of EB1. Purified GST-EB1 deletion mutants were used to isolate GFP-DDA3 from 293T cell lysates. Western blotting with anti-GFP antibodies showed a specific interaction. (e) DDA3 associated with EB1 via its C-terminal domain. Purified GST-EB1 was used as an affinity matrix to isolate GFP-DDA3 and its fragments from the lysates of 293T cells. Western blotting was performed as described in d. (f) Schematic representation and summary of the binding studies for a series of DDA3 deletion mutants in e. +, positive; +/−, weak; −, negative. Numbers indicate positions of the amino acid residues. CC, coiled-coil region.
