Figure 8

HGF-FN Induced Activation of Map Kinase and PI-3 Kinase Pathways is Ras dependent Panel A &B-HMVEC suspensions were pre-treated with buffer (no inhib.) or inhibitors FPT III (100 μM) and control GGTI (2 μM) for 45 min at room temperature prior to stimulation with HGF with no ECM proteins, or HGF plus collagen-1 or HGF-FN complexes for 60 min at room temperature. Cells were pelleted and lysed in an ice-cold lysis buffer. Samples were analysed by SDS-PAGE and Western blotting probing for phospho Erk 1/2 (panel A) and phospho AKT (panel B). The relative band intensities measured by image analysis software have been placed above each band as a ratio of the signal obtained in the sample where no inhibitor was present. Blots were stripped and re-probed with antibodies to Erk 2 and Akt to confirm equal loadings. Panel C-HMVEC suspensions (5 × 10⁶/ml) were incubated with either FN or collagen in the presence or absence of HGF as shown for 60 min at room temperature. Cells were pelleted and lysed in lysis buffer. Immunoprecipitation analyses of the lysates were performed with antibodies to integrins α5β1 and α2β1 and the immune complexes analysed by SDS-PAGE and Western blotting probing with antibodies to Ras. Visualization was by chemiluminescence.