Fig. 4. Culture of HT-29 cells on laminin 5 triggers E-cadherin accumulation at cell-cell contacts and colocalization with actin. (A) Western blot analysis of E-cadherin, p120^ctn and ß-catenin distribution in Triton-soluble fractions (TS) and Triton-insoluble fractions (TI) obtained from lysates of HT-29 cells cultured in standard medium (HT-29) or on laminin 5 matrix for 24 hours (HT-29 LN 5). Histograms showing the percentage of molecules associated with the TI fraction were determined by densitometry of the blots visualized by chemiluminescence (Amersham ECL reagents) using the Image Master VDS-CL (Amersham-Pharmacia Biotech) imaging device. The data were compiled from five independent experiments; error bars represent mean ± s.e.m. The proportion of E-cadherin and ß-catenin associated with the cytoskeleton is increased in HT-29 cells cultured on laminin 5 matrix. (B) Reconstituted images along the Z axis from a series of optical sections. HT-29 cells grown in standard medium (left panels, HT-29) or on laminin 5 matrix for 24 hours (right panels, HT-29 LN 5) were immunostained with anti-E-cadherin (green) and TRITC-conjugated phalloidin (red), and analyzed by confocal microscopy as described under the Materials and Methods. In HT-29 LN 5 cells, E-cadherin was localized to the apicolateral membrane in association with the actin cytoskeleton. Bars, 16 µm.