FIGURE 4.

Detection of αE-catenin·GFP variants, α18-specific epitope, and vinculin in transfected DLD-1-R2/7 cells and their effect on E-cadherin-mediated cell adhesion strength. A, localization of GFP (left panels), vinculin (middle left panels), and α18 epitope (middle right panels) in cells transiently transfected to express αEcatGFP (top panels), aa1–510 αEcatGFP mutant (middle panels), or αEcatMutVincGFP mutant (bottom panels). Merged images are shown in the right panels. Scale bar, 10 μm. Arrows point to some of the intercellular adhesions between transfected cells. B, mean ratio between vinculin and catenin (GFP) (vinc./αcat, blue bars) intensity and α18 and catenin (GFP) intensity (α18/αcat, pink bars) were measured on confocal images at the cell contact area of DLD-1-R2/7 cells transiently transfected to express either αEcatGFP or the mutants aa1–510 αEcatGFP or αEcatMutVincGFP. The number of cell-cell contacts analyzed was 12, 14, and 11, respectively. Error bars, S.E. ***, p < 0.001 (two-tailed) by comparing with the values of cells expressing αEcatGFP. C, mean SF measured at 4 min of contact for αE-catenin-depleted cells transiently transfected to express the αEcatGFP (black bar), αEcatmutVincGFP mutant (blue bar), or aa1–510 αEcatGFP mutant (red bar). Error bars, S.E.M. ***, p < 0.001 (two-tailed). D, Western blot analysis of lysates of flow cytometry-sorted GFP⁺DLD-1-R2/7 cells transfected to express αEcatGFP, αEcatmutVincGFP, and aa1–510 αEcatGFP (see “Experimental Procedures”).