GAS6 modulates VEGF-A–induced EC chemotaxis through activation of Axl. (A) EC chemotaxis was evaluated in presence of GAS6 alone (♦) or associated with VEGF-A₁₆₅ (•) or VEGF-A₁₂₁ (▪) (10 ng/mL). Results of 1 experiment performed in triplicate representative of 3 independent experiments are shown. Data are presented as percentage of inhibition of VEGF-A₁₆₅ (403 ± 34 cells/microscopic field) or VEGF-A₁₂₁ (389 ± 24 cells/microscopic field) induced chemotaxis. Results were analyzed by 1-way analysis of variance (ANOVA) (F = 287.28) and Student-Newman-Keuls test. *P < .05 versus stimulated cells in absence of GAS6. (B) Chemotaxis of ECs in presence of Ab anti–Axl-N or Axl-Ig. Results of 1 experiment performed in triplicate representative of 4 independent experiments are shown. Data were analyzed by ANOVA (F = 7.93) and Student-Newman-Keuls test. *P < .05 versus GAS6 (1 ng/mL) alone; **P < .05 versus GAS6 + anti–Axl-N; §P < .05 versus GAS6 + Axl-Ig. (C) Effect of GAS6 (1 ng/mL) on FGF-2 (20 ng/mL) and HGF (10 ng/mL) induced EC chemotaxis. □ indicates absence, ▪ presence of GAS6. (D) Axl phosphorylation by GAS6. Quiescent, confluent ECs were stimulated for 30 minutes as indicated at 37°C with 5% CO₂. Cells were lysated and immunoprecipitated for 2 hours at 4°C with an anti-Axl Ab. Immunoprecipitate was analyzed by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with anti–P-Tyr mAb or an anti-Axl Ab. Immunoreactive bands were detected by enhanced chemiluminescence technique. I.P. indicates immunoprecipitate; W.B., Western blot. The results are representative of 4 similar experiments. Error bars in A-C indicate standard deviation.