CD44v6 controls Met internalization.

<p>(<b>A</b>) Left side: HeLa cells were transfected either with a pool of control siRNAs or a mixture of two different CD44v6 specific siRNAs and starved for 24 hours (Material and Methods). The cells were biotinylated (0,5 mg/ml), induced with HGF (50 ng/ml) and treated with MESNA. After cell lysis, proteins were pulled down with a NeutrAvidin resin and subjected to Western Blot analysis with Met or the Transferrin receptor (TfR) antibodies. For the first sample the cells were kept at 4°C. –M: the cells were not treated with MESNA. Right side: Western Blot analysis of cell lysates of ctrl siRNAs and CD44v6 siRNAs transfected HeLa cells using the CD44v6 and the TfR antibodies. (<b>B</b>) Starved HeLa cells respectively HT29 cells were incubated with the v6 peptide or a control peptide for 10 minutes at 37°C and then induced with 25 ng/ml of HGF for the indicated time points. Cells were then either lysed and the lysates were subjected to Western Blot analysis for phospho-Met and Met (below) or cells were fixed, permeabilized and stained for Met with specific antibodies (red) (above). Nuclei were stained with Dapi and images were taken with a confocal microscope (Leica SPE) using a 63× objective. The quantification of three independent experiments (n = 40) is shown. The percentage of cells with Met exclusively located at the plasma membrane was calculated for each time point. Student´s t test: ***p<0,001.