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Fig. 6. PI 3-kinase inhibition interferes with E-cadherin expression and its association with the cytoskeleton. (A) HT-29 cells grown for 24 hours on laminin 5 matrix (LN 5) were treated with increasing concentrations of LY294002 as indicated and then lyzed. Whole-cell lysates were separated by SDS-PAGE and subjected to immunoblotting for E-cadherin. Expression of actin was used for normalization. *P<0.05; **P<0.01, as determined by Student's t-test. (B) Upper panel: whole lysates of HT-29 cells expressing the vector alone (pBabe) or 
p85 were subjected to immunoblotting for p85. Expression of tubulin was used as a loading control. Lower panel: HT-29 cells expressing the vector alone (pBabe) or p85 grown on plastic or laminin 5 matrix for 24 hours were lyzed, then analyzed for E-cadherin expression by western blotting. The band densitometry was normalized with tubulin to allow quantification. The results are representative of three independent experiments. (C) Upper panel: HT-29 cells grown for 24 hours on plastic (PL) or laminin 5 matrix (LN 5) treated with (+) or without (-) 50 µM LY294002 were lyzed and then the Triton-soluble (TS) fraction and the Triton-insoluble (TI) fraction (i.e. cytoskeleton-associated proteins) were separated by SDS-PAGE and immunoblotted for E-cadherin. The results are representative of at least three independent experiments. Lower panel: HT-29 cells expressing vector alone (pBabe) or p85 were grown on plastic or laminin 5 matrix for 24 hours. E-cadherin and F-actin were detected by immunofluorescence using HECD1 antibody and TRITC-conjugated phalloidin, respectively. Optical sections were performed using a confocal microscope. Bars, 50 µm.
