TAM receptor expression and siRNA knockdown validation. (A) Tyro3, Axl, and Mer gene transcript levels relative to that of GAPDH as determined by quantitative real-time PCR after 24 and 48 hours of EC culture in growth factor–containing medium. (B-C) The effectiveness and the specificity of targeted siRNA in silencing Tyro3, Axl, and Mer gene expression, real-time quantitative PCR 24 hours after siRNA transfection (B) or Western blot analysis 24 hours after siRNA transfection (C). EC cultures were transfected with either the specified siRNA used at 5nM for Tyro3 and Axl or at 50nM for Mer or with vehicle (5 μL/well siPORT) or as a control with 50nM nontargeting siRNA. For panel B, cells were harvested 24 hours after transfection and total RNA extracted. Reverse transcription was performed using 2 μg of total extracted RNA. Tyro3, Axl, and Mer gene expression was assessed relatively to that of GAPDH by quantitative real-time PCR and is expressed as percentage of the same ratio in siPORT-transfected ECs. Data were obtained from 3 independent experiments each in triplicate ± SD. ***P < .001. **P < .01. *P < .05. ns indicates not significant. For panel C, equivalent amounts of protein form each sample were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-Mer, anti-Axl, or anti-GAPDH antibodies.