Effect of peptide 12 on α5β1 integrin-mediated downstream signaling. HUVECs were starved and treated with cycloheximide as described in “Western blotting analysis” and then plated on peptide 12-, or fibronectin- (FN)-coated dishes in the presence or absence of VEGF-A. (A) Immunoblotting analysis for the presence of phosphorylated-FAK or phosphorylated-ERK1/2 polypeptides after 1-hour adhesion. Actin staining was performed to normalize the results (ACT). (B) Histograms showing the relative OD of the bands in panel A, normalized using absorbance of the actin bands for each sample, after subtraction of the background (phosphorylation levels in cells plated on BSA-coated dishes). (C) Immunoblotting analysis of phosphorylated-Shc and total Shc polypeptide after 30 minutes of adhesion. The histogram shows the OD ratio between the phosphorylated band and the total protein. Data represent the results of a representative experiment.