VEGF-A and FGF2 signaling in Sema6A-silenced endothelial cells. (A) Control and Sema6A-silenced HUVECs were incubated 15 minutes with VEGF-A (100 ng/mL). Cell lysates were tested for phosphorylated and total VEGFR2, AKT, and ERK1/2; β-actin staining was used as a loading control. (B) Intracellular phospho-AKT and (C) intracellular phospho-ERK were analyzed by flow cytometry under the same experimental conditions of A. The results represent the relative percentage of phospho-AKT and phosphor-ERK positive cells (representative experiments). (D) FGFR1 gene expression was measured by quantitative PCR in Sema6A-silenced and control HUVECs. The results reflect the mean (± SEM; n = 6) mRNA levels relative to control. (E) Sema6A-silenced and control HUVECs were incubated (15 minutes) with FGF2 (100 ng/mL). Phosphorylation levels of AKT and ERK1/2 were assessed in the cell lysates by Western blotting. (F) Intracellular phospho-AKT and (G) intracellular phospho-ERK were analyzed by flow cytometry under the same experimental conditions of E.