Forced expression of VEGFR3 in VEGFR2⁺ cells stimulates VEGF-induced endothelial differentiation. (A) A scheme showing transfection and in vitro differentiation of mouse ES cells, MGZ-5. See “Materials and methods” for details. (B-C) MGZ-5 cells were transfected with mock, VEGFR3 wild-type (WT) and kinase-negative mutant (G857R and R1041P) cDNAs. Transfected cells were selected by puromycin and differentiated into mesodermal cells. Expression levels of these constructs were determined by immunoblot analysis using an anti-VEGFR3 antibody (B). The differentiated cells were sorted using an anti-VEGFR2 antibody. Expression levels of VEGFR3(WT) in unsorted (Total), VEGFR2⁺ and VEGFR2^- cells were determined (C). α-tubulin was used as a loading control in panels B and C. (D) MGZ-5 cells transfected with mock, VEGFR3 (WT), and kinase-negative mutants (G857R and R1041P) were differentiated into mesodermal cells and sorted using an anti-VEGFR2 antibody. VEGFR2⁺ cells were cultured in differentiation medium with or without VEGF-A (40 ng/mL) and VEGF-C (400 ng/mL). After 4 days, cells were doubly stained with anti–PECAM-1 (purple) and anti-SMA (brown) antibodies. Numbers of ECs and total cells were counted and shown as ECs/total (%). Scale bar, 100 μm. (E) HEK293 cells were transfected with mock, VEGFR3 (WT), and kinase-negative mutant (G857R and R1041P) cDNAs. Transfected cells were treated with or without VEGF-A (40 ng/mL), VEGF-C (400 ng/mL), and VEGF-D (500 ng/mL) for 20 minutes. Cell lysates were immunoprecipitated by anti-VEGFR3 antibody, followed by immunoblotting using anti-phosphotyrosine (P-Tyr, top panel) and VEGFR3 (bottom panel) antibodies.