Expression of SHP-2 C/S mutant inhibits GAS6 modulation of EC chemotaxis induced by VEGF-A₁₆₅. (A) Effect of SHP-2 C/S expression on Tyr phosphorylation of SHP-2. Quiescent, confluent infected ECs were stimulated for 30 minutes with GAS6 (1 ng/mL). Cell lysates were immunoprecipitated with Ab anti–SHP-2 and immunoblotted with the same Ab or with Ab anti–P-Tyr. (B) Effect of SHP-2 C/S expression on Tyr phosphorylation of Axl. Pinco-ECs and SHP-2 C/S-ECs were stimulated with GAS6 (1 ng/mL for 30 minutes). Immunoprecipitation was performed with Ab anti-Axl, and proteins were immunodetected with Ab anti–P-Tyr or anti-Axl. (C) Effect of SHP-2 C/S expression on Tyr phosphorylation of VEGFR-2. Pinco-ECs and SHP-2 C/S-ECs were preincubated with GAS6 (1 ng/mL) for 20 minutes and consequently stimulated for 10 minutes with VEGF-A₁₆₅ (10 ng/mL). Immunoprecipitation was performed with Ab anti–VEGFR-2, and proteins were immunodetected with Ab anti–P-Tyr or anti–VEGFR-2. (D) EC chemotaxis induced by VEGF-A₁₆₅ and VEGF-A₁₂₁ (both at 10 ng/mL) was evaluated in cells infected with vector alone, or carrying SHP-2 C/S mutant or SHP-2. When indicated 1 ng/mL GAS6 was added. Results of 1 experiment performed in triplicate representative of 3 independent experiments are shown. Data were analyzed by ANOVA (F = 495.89 for SHP-2, F = 876.7 for SHP-2 C/S) and Student-Newman-Keuls test. *P < .05 versus control; §P < .05 versus VEGF-A₁₆₅ or VEGF-A₁₂₁ alone.