Biomolecules & Therapeutics : eISSN 2005-4483 / pISSN 1976-9148

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Fig. 3. NOEY2-N directly binds with VEGFR-2 but not VEGFR-1 and reduces VEGF-induced VEGFR-2 phosphorylation (Tyr-1175). (A) Positive interactions were validated by monitoring cell growth on a medium lacking leucine (upper panel) and forming blue colonies (lower panel) on X-gal plates, including 2% galactose. β-galactosidase activity (unit), evaluated by adding ONPG, is presented below the corresponding lanes. Data are presented as mean ± SD from three independent experiments. (B) Co-IP of NOEY2-N with VEGFR-1 or VEGFR-2. Immunoprecipitation (IP) was performed using anti-Flag antibodies in lysates from transfected HEK293T cells, followed by western blotting with anti-NOEY2, anti-VEGFR-1, and anti-VEGFR-2 antibodies. (Left panel) lane 2, pcDNA3.1 (expression vector only) and pcDNA3.1/Flag-NOEY2-N transfectant; lane 3, pcDNA3.1/Flag-NOEY2-N and pcDNA3.1-VEGFR-1 transfectant. (Right panel) lane 2, pcDNA3.1 (expression vector only) and pcDNA3.1/Flag-NOEY2-N transfectant; lane 3, pcDNA3.1/Flag-NOEY2-N and pcDNA3.1-VEGFR-2 transfectant. (C) SKOV-3 cells were treated with VEGF and then control-transfected or transfected with NOEY2-N. NOEY2-N significantly reduced phosphorylation of VEGFR-2 (Tyr-1175) triggered by VEGF. Phosphorylation of VEGFR-2 (Tyr-951 or Tyr-1175) was assessed by the specific antibody. VEGFR-2 was used as the loading control. (D) Inhibitory effect of VEGFR-2-dependent transcription by NOEY2-N. SKOV-3 and HUVECs were co-transfected with 500 ng of a VEGFR-2 expression plasmid (pcDNA3.1/VEGFR-2), 500 ng of VEGFR-2-Luc, and increasing concentrations of plasmid-encoding NOEY2-N (pcDNA3.1/Flag-NOEY2-N) (50, 250 and 500 ng). Data are presented as mean ± SD from three independent experiments. *p<0.05 compared to control. (E) SKOV-3 cells were incubated with/without 10 ng/mL VEGF stimulator and were then control (expression vector only)-transfected or transfected with NOEY2-N. Expression levels of VEGF and HIF-1α were then visualised by immunoblotting. Experiments were repeated at least three times with similar results. (F) SKOV-3 cells were seeded to a confluence of 80%. Exponentially growing cells were transfected with increased concentrations of NOEY2-N (0.0-1.2 μg). Expression levels of VEGF were calculated by scanning densitometry and normalised to levels of loading control. Each data point represents triplicate samples, and bars indicate mean ± SD. *p<0.05 compared to control.
Biomolecules & Therapeutics 2021;29:506~518 https://doi.org/10.4062/biomolther.2021.121
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