VEGF-C(C152S) did not stimulate the differentiation of VEGFR3-transfected VEGFR2⁺ cells into ECs. (A) HEK293 cells were transfected with mock and VEGFR3 cDNAs (long and short forms; L and S, respectively). Transfected cells were treated with or without VEGF-A or the supernatant (SFO3) of HEK293 cells transfected with VEGF-C(WT) and (C152S) cDNAs for 20 minutes. Cell lysates were immunoprecipitated by anti-VEGFR3 antibody, followed by immunoblotting using antiphosphotyrosine (P-Tyr, top) and VEGFR3 (bottom) antibodies. Top blot is phosphorylated VEGFR3; bottom blot, VEGFR3. A indicates VEGF-A; WT, sVEGF-C(WT); 152, sVEGF-C(C152S). (B) VEGFR2⁺ cells transfected with mock and VEGFR3 (L and S) were cultured in SFO3 serum-free medium in the presence of a supernatant of HEK293 cells as described in panel A. After 2 days, cells were doubly stained with anti–PECAM-1 (purple) and anti-SMA (brown) antibodies. Scale bar, 100 μm. (C) RT-PCR analysis of vascular and lymphatic endothelial markers in the differentiated ECs described in panel B. M indicates mock; L, long; S, short.