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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 6: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability.

Figure 6:

(A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, 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 immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. (B) JAK2 phosphorylates STAT3 in vitro. In vitro kinase assay was performed using purified human STAT3 protein and kinase active JAK2 protein. (C) Images of footpads from C57BL/6 wildtype mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans blue, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle were injected into the root of the footpad. After 30 minutes, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans blue leakage in C57BL mice treated with vehicle or AG490. n=4 mice per group. Each mouse was injected with PBS on right anterior and posterior footpads and VEGF on left anterior and posterior footpads. **P<0.01, ***P<0.001, paired t-test.