Tara-KD–dependent regulation of E-cadherin expression and Rac1 activation. (A) Immunoblotting of control MDCK and Tara-KD cells for E-cadherin, cadherin-6, and α-catenin at 24 and 48 h after EDTA/trypsin treatment and replating. Note the marked decrease in the protein level of E-cadherin by Tara-KD. The protein level of cadherin-6 was increased by Tara-KD compared with control cells. Each of E-cadherin, cadherin-6, Tara, and α-catenin was probed by its antibody in the presence of the loading control anti–α-tub at the same time by mixing antibodies. (B and C) Semi-quantitative RT-PCR (B) and luciferase reporter assays (C) for the effect of Tara-KD on E-cadherin mRNA levels and promoter activity, respectively. Note that the E-cadherin mRNA and its promoter activity, respectively, decreased significantly at 48 h after cells were seeded (B and C), whereas there was no obvious change in the cadherin-6 mRNA level (not depicted). The results are expressed as means ± SD (error bars) and are representative of four independent experiments. **, P < 0.01; *, P < 0.05. (D) Effect of Tara-KD on the level of active Rac-1 determined by a PAK pull-down assay. The Rac1 activity was increased in Tara-KD cells 24 h after replating. The quantification of the immunoblotted signals revealed the significant changes of Rac1 activation by Tara-KD at 24 and 48 h after being seeded (see the right panel). ND, not detected. Densitometric quantification of Western blot bands was performed using Photoshop 7.0 (Adobe). As to the “Relative Intensity,” the ratio of intensity of GTP-Rac1 to total-Rac in control was normalized to 1.0. The results are expressed as means ± SD (error bars) and are representative of three independent experiments. **, P < 0.01; *, P < 0.05. (E) FRET analysis of Rac activation. At 12 and 24 h after replating, the Rac1 activation was detected at cell–cell contacts in both control and Tara-KD cells. At 48 h, Rac activation was still detectable in Tara-KD cells but not in control cells. Bar, 10 µm.