HGF-FN and HGF-VN complexes augment HMVEC migration via the PI3 kinase pathway. Panel A-HMVEC suspensions (1 × 106/ml) were stimulated with HGF (10 ng/ml) in the absence of ECM molecules or in the presence of collagen-1, FN or VN (2 μg/ml) for varying time intervals (2–120 min) at room temperature. The reaction was stopped by rapid centrifugation and lysis of the cell pellet. Samples of cell lysate were analysed by SDS-PAGE and Western blotting probing with an antibody with specificity for phosphorylated Erk 1/2 (top panel). The blots were reprobed with an antibody to Erk 2 (bottom panel). Panel B-The samples from panel A were also probed simultaneously with an antibody with specificity for phosphorylated Akt (top panel) and these blots were stripped and re-probed with an antibody to Akt. Visualization was by chemiluminescence. Panel C &D-Effect of inhibitors on HMVEC migration in response to HGF-FN and HGF-VN respectively. The data is a representative experiment using triplicate samples which was performed three times giving essentially similar results.