Figure 5

Cilengitide inhibits FAK and Src but not VEGF-induced phosphorylation of KDR and ERK1/2 in endothelial cells. (A) Western blotting and (B) densitometric analysis of FAK, Src and Erk1/2 in HUVEC. Cells were maintained 24 hours in serum-free medium on uncoated dishes and then treated with cilengitide for 1 hour at concentrations indicated. Cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane to detect phosphorylated FAK, Src and Erk1/2. (C) Western blotting analysis of FAK, KDR and Erk1/2 and (D) densitometric analysis of FAK and Erk1/2 in PAE-KDR cells. Cells were maintained 24 hours in serum-free medium, treated with VEGF (5 ng/ml) and cilengitide at concentrations indicated and incubated 30 minutes on ice and 7 minutes at 37°C. For detection of FAK and Erk1/2, cell lysates containing same amount of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. For detection of KDR, cells were extracted and subjected to KDR immunoprecipitation. Total proteins from immunoprecipitates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Thereafter, membranes were probed with specific antibodies against phosphorylated FAK, KDR and Erk1/2.