Fig. 7. PI 3-kinase associates with E-cadherin complexes upon cell adhesion on laminin 5. Upper panel: western blot analysis of the distribution of the p85 regulatory subunit of PI 3-kinase. HT-29 cells grown for 24 hours on plastic (PL) or laminin 5 matrix (LN 5) were fractionated as described under the Materials and Methods, to separate nuclear-, cytosolic- and membrane-enriched fractions, then analyzed by western blot using an anti-p85 antibody. The blot shown is representative of four independent experiments. Middle panel: HT-29 cells grown for 24 hours on plastic (PL) or laminin 5 matrix (LN 5) were immunostained with anti-E-cadherin (green) and anti-p85 (red) antibodies and analyzed by confocal microscopy on optical sections. Bar, 30 µm. Lower panel: the p85 subunit of PI 3-kinase was immunoprecipitated (IP) from 500 µg of proteins from whole-cell lysates of HT-29 cells cultured for 24 hours on plastic or laminin 5 matrix. Proteins of the immunoprecipitates were solubilized into Laemmli's buffer and resolved by SDS-PAGE before western blotting (WB) to determine the amount of PI 3-kinase and E-cadherin in the immunoprecipitates. The blot shown is representative of three independent experiments.