Accumulation of lipid rafts at the cell-substratum interface upon cell adhesion. (a) A431 cells were fractionated as described in Methods. An equal portion of cell lysates from soluble and insoluble fractions was analysed by immunoblotting with antibodies as indicated. Flotillin1 is used as a marker for lipid rafts. ERK is used as a marker for non-rafts. The phosphorylation of Met and EGFR was measured. Data shown are representative from two independent experiments. (b) A431 cells were kept in suspension for 30 min and replated on glass coverslips coated with 100 μg/ml poly-L-lysine for various time as indicated. The cells were stained for lipid rafts with Alexa 488-conjugated cholera toxin B subunit and visualized by confocal microscopy. Representative XZ and XY images at indicated time are shown. The XY sections were taken at the height of 3.5 μm above the bottom. Scale bar, 5 μm. (c) A431 cells were replated on glass coverslips coated with 100 μg/ml poly-L-lysine for 30 min, stained for lipid rafts with Alexa 488-conjugated cholera toxin B subunit, and visualized by total internal reflection fluorescence (TIRF) microscopy. The morphology of the same cell was taken by a differential interference contrast (DIC) objective. Scale Bar, 5 μm.