CD44v6 controls Met internalization.

(A) 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) 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.