RAF1^S259A induces SEMA6A and NTNG1 expression. (A) qPCR analysis of guidance signaling pathways in HUVECs infected with control, WT RAF1, or RAF1^S259A lentiviruses. Data represent mean ± SEM of 4 independent experiments. *P < .05; **P < .01. (B) Effect of MEK and PI3K inhibition on SEMA6A and NTNG1 expression. HUVEC transduced with control, WT RAF1, or RAF^1S259A lentiviruses were treated with DMSO, MEK inhibitor U0126 (10 mM) for 24 hours. Gene expression was assessed by qPCR. Data represent mean ± SEM of 3 independent experiments. (C) qPCR analysis of guidance signaling pathways in primary ECs isolated from E12.5 embryos. n = 5 embryos. (D) Effect of NOTCH inhibition on SEMA6A and NTNG1 gene expression was assessed by qPCR. HUVECs transduced with control, WT RAF1, or RAF1^S259A lentiviruses were treated with DMSO or DAPT for 24 hours. Data represent mean ± SEM of 3 independent experiments. (E) Immunoblot of HUVECs treated with DAPT.