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[Preprint]. 2020 Nov 20:2020.10.27.358374. Originally published 2020 Oct 28. [Version 3] doi: 10.1101/2020.10.27.358374

Figure 1: VEGF/VEGFR-2 induces STAT3 phosphorylation and nuclear localization.

Figure 1:

(A) Stimulation of HUVEC with 25ng/ml recombinant human VEGF-165 protein for 2 and 5 minutes induces p-VEGFR-2 (Y1175) and p-STAT3 (Y705) via immunoblotting. (B) VEGF (25 ng/ml) stimulation in HUVEC for 10 and 30 minutes promotes co-immunoprecipitation of STAT3 and VEGFR-2. (C) VEGF (25 ng/ml) stimulation in HUVEC for 10 and 30 minutes promotes co-immunoprecipitation of p-STAT3 (Y705) and p-VEGFR-2 (Y1175). (D) GST pull-down of VEGFR-2 with STAT3. Lysates of HUVEC stimulated with serum for 30 minutes were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblot. GST-STAT3 and GST were both detected using an anti-GST antibody. (E) VEGF stimulation for 2 minutes and 5 minutes promotes nuclear localization of STAT3. DAPI is blue. Scale bar, 20 μm. (F) Quantification of nuclear immunofluorescence staining intensity. Mean ±SEM, one-way ANOVA. *P<0.05, ****<0.0001. (A–E) Images are representative of multiple biological replicates.