Control of VEGF signaling by LEC-fate regulators, Prox1 and COUP-TFII. (A) Western blot analyses show that knockdown of Prox1 and/or COUP-TFII up-regulates the expression of VEGFR-2 and/or NP-1 and activates phosphorylation of VEGFR-2 (Y1175). (B) Chromatin immunoprecipitation assays demonstrate that, although Prox1 protein is associated with the proximal promoters of VEGFR-2 and NP-1, COUP-TFII protein is associated only with the VEGFR-2 promoter. (C-D) Repression of the VEGFR-2 proximal promoter by Prox1 (C) or COUP-TFII (D). A series of VEGFR-2 promoter constructs was transiently transfected into HEK293 cells for 48 hours with a control (CTR), wild-type Prox1 (Prox1WT), or DNA-binding mutant (Prox1Mut) vector. Each number (eg, −716) represents the distal end of human VEGFR-2 promoters, and all of them end at +268.³⁰  Only 10% of luciferase activity was charted for pGL2 (pGL2-Basic vector) because of low activities of VEGFR-2 promoters. Data are relative luciferase activity ± SD. (E) Prox1 and COUP-TFII recombinant proteins bind to the VEGFR-2 proximal promoter. EMSA was performed against a DNA probe with sequences between −90 and −51 nucleotides of human VEGFR-2 promoter³⁰  using recombinant His-Prox1, GST, or GST-COUP-TFII protein. (F) Up-regulation of NP-1 and VEGFR-2 by activated Notch receptor in LECs. Western blot analyses show the Notch-mediated regulation of Prox1, COUP-TFII, NP-1, and VEGFR-2.