Fig. 5. Laminin 5-induced activation of PI 3-kinase in HT-29 cells. (A) Effect of different ECM proteins on PI 3-kinase activity. HT-29 cells were allowed to adhere for 24 hours to dishes uncoated (PL) or coated with purified laminin 5 (LN 5), collagen IV (CO IV), or fibronectin (FN) at 10 µg/ml. To test the specificity of the interaction with laminin 5, HT-29 cells were preincubated with the anti-3 chain of laminin 5 BM165 at a concentration of 20 µg/ml and then allowed to adhere to laminin 5. (B) Time course of PI 3-kinase activation by laminin 5 matrix. HT-29 cells were allowed to adhere for 1, 4, 24 or 48 hours to dishes coated with laminin 5 matrix. In (A) and (B), total cell lysates were prepared and equivalent amounts of proteins were used in immunoprecipitation and kinase assays. Western blots performed on immunoprecipitated p85 subunit were carried out as controls. Phosphorylated lipids were resolved by thin layer chromatography. The amount of radiolabeled PtdIns(3,4,5)P₃ (PIP3) was determined for each condition by densitometry with ImageQuant software. Data represent the average of percentage of control activity (mean ± s.e.m. of three to four independent experiments) and one representative chromatogram is shown. ^*P<0.05; ^**P<0.01, as determined by Student's t-test. (C) HT-29 cells were incubated with 2 mM EGTA or 10 µg/ml of integrin or E-cadherin-specific antibodies and allowed to adhere to plates coated with laminin 5 matrix for 24 hours. mAbs specific for ₃ (P1B5), ₆ (GoH3) or ß₁ (P4C10) integrin subunits and E-cadherin (HECD1) were used at 10 µg/ml. Total cell lysates were prepared and equivalent amounts of proteins were subjected to immunoprecipitation and kinase assays as described previously. ^**P<0.01, as determined by Student's t-test.