Involvement of Trio in Tara-KD–dependent regulation of E-cadherin expression and Rac1 activation. (A) Schematic drawing of the Tara deletion constructs. (B) The Tara domain responsible for regulating the activation of Rac1. The mid-domain of Tara was sufficient to cause the down-regulation of Rac1 activation in Tara-KD cells. (C) Analysis of the Tara domain responsible for the restoration of E-cadherin in Tara-KD cells, by transfection with Tara deletion mutants. Consistent with the Rac1 activity, the mid-domain of Tara was essential to rescue the expression of E-cadherin in Tara-KD cells. (D) Effect of the Trio RhoGEF inhibitor ITX3 on the activation level of Rac1 in Tara-KD cells. In the presence of 100 µM ITX3, the Rac1 activation in Tara-KD cells was markedly decreased, to the level in control cells. The results are expressed as means ± SD (error bars) and are representative of three independent experiments. **, P < 0.01; *, P < 0.05. Densitometric quantification of Western blot bands was performed using Photoshop 7.0 (Adobe). As to the “Relative Intensity,” the ratio of intensities of E-cadherin to α-tubulin and cadherin-6 to α-tubulin in control were normalized to 1.0. (E) Effect of ITX3 on the expression of E-cadherin and cadherin-6 in Tara-KD cells. ITX3 blocked the Tara-KD–induced down-regulation of E-cadherin and up-regulation of cadherin-6 in a dose-dependent manner. The right panels show the quantification of the immunoblotting data (as shown in Fig. S4 C). The results are expressed as means ± SD (error bars) and are representative of three independent experiments, in which antigens were immunoblotted in the same membranes. **, P < 0.01; *, P < 0.05.