Fig. 4.

Effects of 3AOA on expressions of VEGFR-1 and VEGFR-2, and activation of VEGFR-1, VEGFR-2 and lymphangiogenesis related downstream signaling factors in rhVEGF-A-treated HLMECs. a-b Expression levels of VEGFR-1 and -2 proteins were determined using Western blot analysis. Amounts of VEGFR-1 and -2 obtained in three independent experiments were quantified and represented as a bar diagram. Levels of VEGFR-1 and -2 in 3AOA- and rhVEGF-A-untreated cells were estimated as 100%. c-d, Cell lysates were immunoprecipitated with anti-phospho-Tyr (anti-p-Tyr). The level of phosphorylated VEGFR-1 and -2 in immunoprecipitates was detected using Western blot analysis with anti-VEGFR-1 and anti-VEGFR-2. Phosphorylation levels of VEGFR-1 and -2 obtained in three independent experiments were quantified and represented as a bar diagram. Phosphorylation levels of VEGFR-1 and -2 in 3AOA- and rhVEGF-A-untreated cells were estimated as 100%. e, HLMECs were serum starved for 6 h, then were treated with different concentrations of 3AOA (0, 2.5, 5 μM) in the presence of rhVEGF-A (20 ng/mL) for 60 min. The phosphorylation levels of FAK, PI3K, AKT, and ERK1/2 were determined using Western blot analysis with anti-p-FAK, anti-p-PI3K, anti-p-AKT, and anti-p-ERK1/2. Data are presented as a mean ± S.D. of three independent experiments (^*p < 0.05, ^**p < 0.01, ^***p < 0.001)