Fig. 3.

Anastellin has no effect on VEGF₁₂₁-induced VEGFR2 activation and signaling. (A) Serum-starved microvessel cell monolayers were pretreated for 60 minutes with increasing concentrations of anastellin, followed by stimulation with either 10 ng/ml VEGF₁₂₁ or VEGF₁₆₅ for 6 minutes. Cell lysates were analyzed by western blotting for changes in the phosphorylation of VEGFR2 and ERK. Total levels of VEGFR2 served as a loading control. (B) Blots were quantified using ImageJ software. Data show the mean±s.e.m. (three independent experiments). ‘C’, untreated control. (C) Microvessel cell monolayers were pretreated with 20 µM anastellin for 60 minutes prior to stimulation with 10 ng/ml VEGF₁₆₅ or VEGF₁₂₁ for 15 minutes. NRP1 was immunoprecipitated (IP) from all lysates, and precipitates were analyzed by western blotting for the presence of VEGFR2. Input amounts of VEGFR2 and NRP1 present in lysates prior to immunoprecipitation are shown on the left.