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. 2011 Dec 2;6(12):e28394. doi: 10.1371/journal.pone.0028394

Figure 2. Mapping the interacting domains of Mixl1 and Brachyury.

Figure 2

(A) Schematic of Brachyury (T) and its truncated mutants. Amino acid residues present in each protein are shown. DNA binding domain (DBD), Transactivation domain (TAD), Repression domain (RD). (B) The amino terminal DBD (T-box) domain of T and a carboxy terminal region can independently interact with Mixl1. 293T cells were transfected with HA mMixl1 and GST-T or its truncated mutants as indicated. GST-T proteins were isolated from whole cell extracts using glutathione resin and the bound fraction analysed by Western blot with an anti-HA antibody. Expression of each protein was confirmed with anti-GST and anti-HA antibodies. (C) Schematic of Mixl1 and its truncated mutants. Amino acid residues present in each protein are indicated. Proline rich domain (Pro), Homeodomain (HD), Activation domain (AD). (D) The Mixl1 HD and amino terminal flanking sequences are required for its interaction with T. 293T cells were transfected with HA mT and GST-Mixl1 or its truncated mutants as indicated. GST-Mixl1 proteins were isolated from whole cell extracts using glutathione resin and the bound fractions were analysed by Western blot with an anti-HA antibody. Expression of each protein was confirmed with anti-GST and anti-HA antibodies. (E) The Brachyury T-box domain and Mixl1 HD associate. Recombinant His mT-box was incubated with recombinant GST, GST mMixl1 or GST mMixl1 HD proteins pre-adsorbed to the glutathione resin. The bound fractions were analysed by Western blot with anti-T N19 and anti-GST antibodies.