Cells were plated sparsely on glass coverslips and incubated for 2 d before immunofluorescent labeling. Cells were washed once with PBS, then fixed in 3% PFA for 30 min. Fixed cells were washed with PBS/10 mM glycine twice and permeabilized in 0.2% Triton X-100/PBS for 5 min. Cells were again washed in PBS/10 mM glycine and blocked in 3% milk/PBS before staining. Primary antibodies mAb pp120 (Transduction Laboratories), anti-β-catenin C-2206 (Sigma-Aldrich), anti-α-catenin C-2081 (Sigma-Aldrich), anti-E-cadherin C-20820 (Transduction Laboratories), and HECD-1 (a gift from Masatoshi Takeichi, Kyoto University, Kyoto, Japan) were used as described previously (Ireton et al., 2002). Other primary antibodies were used as follows: anti-tubulin (DM1a; Sigma-Aldrich) 1:1000, anti-vinculin (hvin-1; Sigma-Aldrich) 1:400, anti-myc (mAb 9E10) 1μg/ml, and SHE78–7 anti E-cadherin (Zymed Laboratories) 1μg/ml. Secondary antibodies goat anti–mouse IgG1 and IgG2a conjugated either to Alexa® 594 or 488 were used at 1.7 μg/ml. Cells were mounted in ProLong Antifade (Molecular Probes, Inc.) according to the manufacturer's instructions and were visualized on a microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) with Immersol 518F oil (Carl Zeiss MicroImaging, Inc.) using a 63×
