Lyn kinase regulates FAK phosphorylation. (A) Effect of Lyn silencing on FAK phosphorylation. HUVECs were transfected with control siRNA or Lyn siRNA. Forty-eight hour after transfection, cells were solubilized, and phosphorylation of FAK was detected by western blotting with rabbit polyclonal antibodies specifically recognizing the phosphorylated FAK residues Tyr³⁹⁷, Tyr^576/577, Tyr⁸⁶¹, or Tyr⁹²⁵. A mouse monoclonal antibody against β-actin was used to verify equal loading. Images are representative of 3 experiments. (B) Lung microvascular endothelial cells isolated from Lyn^−/− and wild-type littermates were cultured and solubilized in SDS sample buffer. Phosphorylation of FAK was detected by western blotting as described above. (C) Effect of expression of constitutively active mutant of Lyn on the phosphorylation of FAK. HUVECs were transfected with a recombinant adenovirus encoding a constitutively active mutant of Lyn kinase (Lyn^Y508F) or null adenovirus (Control) and FAK phosphorylation was analyzed. Twenty-four hours after infection, cells were solubilized, and phosphorylation of FAK was detected by western blotting. (D) Stimulation of FAK phosphorylation by VEGF. HUVECs were incubated with VEGF (200 ng/mL) for various lengths of time. Cells were then solubilized and phosphorylation of FAK was detected by western blotting.