(A) Western blot shows that ERK is activated in S259A primary mouse lung ECs (left panel). ERK activity was quantified by densitometry and is represented as the ratio of pERK1/2 to total ERK1/2 (right panel). Data represent the mean ± SEM of 3 independent experiments. (B) Immunofluorescence staining showing higher pERK1/2 (red, arrowheads) in β-gal–positive (green) ECs of E12.5 S259A embryos. Scale bar: 100 μm. (C) Effect of MEK and PI3K inhibition on lymphatic gene expression. HUVECs transduced with control, wild-type RAF1, or RAF1^S259A lentiviruses were treated with DMSO, MEK inhibitor U0126 (10 μM), or PI3K inhibitor LY294002 (10 μM) for 24 hours. SOX18, PROX1, VEGFR3, and LYVE1 expression was assessed by qPCR. Data represent the mean ± SEM of 3 independent experiments. *P < 0.05; **P < 0.01. (D) Effect of mTOR and AKT inhibition on SOX18, PROX1, and COUP-TFII expression. HUVECs transduced with GFP or RAF1^S259A adenoviruses were treated with DMSO, rapamycin (10 μM), AKT inhibitor VIII (10 μM), or LY294002 (10 μM) for 24 hours. SOX18, PROX1, and COUP-TFII expression was assessed by qPCR. Data represent the mean ± SEM of 3 independent experiments. (E) Constitutive active ERK is able to induce lymphatic genes. HUVECs were transduced with adenovirus expressing lacZ, cytosolic localized constitutive active ERK (ME), or nuclear localized constitutive active ERK (ME-LA). SOX18, PROX1, VEGFR3, LYVE1, and PDPN expression was assessed by qPCR. Data represent the mean ± SEM of 3 independent experiments. jv, jugular vein.