Coreceptor function of CD44v6 in ECs. (A) CD44 variant exon-specific RT-PCR analysis in HUVECs. The method and the primers have been described.^13,25  “s” refers to the use of 2 primers in the CD44 constant exons 5 and 15 (black boxes in the schematic drawing of the relevant parts of the CD44 gene); the other lanes refer to PCRs performed with the forward primers in variant exons and the reverse primer in exon 15. “M” refers to a DNA ladder. The Western blot shows a staining of cell lysates with the CD44v6-specific antibody VFF18. A total of 20 μg of protein was applied to each, except for HT29 (5 μg). Size markers for apparent molecular weight are indicated. HepG2 were used as CD44v6 negative cells, HT29 as positive ones. (B) Signal transduction induced by HGF, VEGF-A165, or PDGF in untreated HUVECs or HUVECs treated with the human v6-specific 14mer peptide or a control peptide. Activation of Erk was measured as described in “Activation of RTKs and Erk.” (C) VEGFR-2 activation by VEGF-A165 in HUVECs was determined after immunoprecipitation of VEGFR-2 and Western blotting with the phospho-specific VEGFR-2 antibody. IgG indicates a control precipitation. Treatment with VEGF-A165 and with the peptides was performed as described in “Activation of RTKs and Erk.” (D) Ligand-induced signaling in HUVECs in the presence of the CD44v6ECD or a mutated version as indicated. Treatments were done as described in “Activation of RTKs and Erk.” The numbers indicate the fold induction as calculated by the computer program ImageJ. All experiments were performed at least 3 times and gave similar results. Vertical lines have been inserted to indicate repositioned gel lanes.