Figure 3
Association of Met with integrins is driven by HGF-FN and HGF-VN complexes. Panel A-HMVEC (1 × 106 /ml) were seeded onto collagen-1, FN or VN coated plastic wells and were stimulated with either saline or HGF (10 ng/ml) for 60 min at room termperature. Cells were lysed and the corresponding lysates (500 μg protein) were immunoprecipitated with antibodies to integrins α2β1 (lanes 1 & 4), αvβ3 (lanes 2 & 5) and α5β1 (lanes 3 & 6). Immunocomplexes were analysed by SDS-PAGE and Western blotting probing with an anti-Met antibody using chemiluminescence detection (top panel). The bottom panel shows the antigen levels of Met in the corresponding cell lysates after analysis by SDS-PAGE (40 μg/lane) and Western blotting probing with an antibody to Met were essentially unaltered during the duration of the experiment. Panel B-HMVEC (5 × 106) were stimulated with HGF or HGF-FN or HGF-VN complexes at the indicated time periods at room temperature. Cells were lysed and immunoprecipitated with a polyclonal antibody to phosphotyrosine Met and the immune complexes analysed by SDS-PAGE and Western blotting using a monoclonal antibody to Met. The Met antigen was detected using chemiluminescence. Panel C-HMVEC (5 × 106) were stimulated with HGF or HGF-FN or HGF-VN complexes for 15 minutes at room temperature. Cells were lysed and the lysates immunoprecipitated with monoclonal antibodies to the integrins α5β1 and αvβ3 and the immune complexes analysed by SDS-PAGE and Western blotting probing with a polyclonal antibody to Met.