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Fig. 5.
JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min 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. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro. In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n=4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.