Figure 3.
Figure 3. Expression of LYVE-1 was induced in VEGFR3-transfected ECs in response to sVEGF-C(WT). (A) HEK293 cells were transfected with VEGFR2 (top) or VEGFR3 (bottom) cDNAs. Transfected cells were treated with or without 400 ng/mL recombinant VEGF-C (rC), sVEGF-C(WT) (sC), and 40 ng/mL VEGF-A (A) for 20 minutes. Cell lysates were immunoprecipitated by anti-VEGFR2 (top panels) or anti-VEGFR3 (bottom panels) antibodies, followed by immunoblotting using antiphosphotyrosine (P-Tyr) antibody. Expression levels of VEGFR2 and VEGFR3 were confirmed by corresponding antibodies. (B) HEK293 cells were transfected with VEGFR2 and VEGFR3 cDNAs. Transfected cells were treated with or without sVEGF-C(WT) for 20 minutes. Cell lysates were immunoprecipitated by anti-VEGFR2 antibody, followed by immunoblotting using antiphosphotyrosine (P-Tyr, top) and anti-VEGFR2 (middle) antibodies. Immunoblotting for total cell lysates was performed using anti-VEGFR3 (bottom) antibody. (C) VEGFR2+ cells transfected with mock and VEGFR3 (long and short forms) cDNAs were cultured in SFO3 serum-free medium in the presence of supernatant (SFO3) of HEK293 cells transfected with mock (i) and VEGF-C cDNA (ii). After 2 days, cells were observed by phase-contrast microscopy (left panels) or doubly stained with anti–PECAM-1 (purple) and anti-SMA (brown) antibodies (right panels). Numbers of ECs and total cells were counted and shown as ECs/total (%). Scale bar, 100 μm. (D) Expression levels of VEGFR3 (long and short forms) in the differentiated ECs were analyzed by immunoblotting using anti-VEGFR3 antibody. Expression levels of PECAM-1 and α-tubulin were confirmed using specific antibodies (lower panels). M indicates mock; L, long; S, short. (E) RT-PCR analysis of lymphatic endothelial markers in the differentiated ECs described in panel C. mVEGFR3 and hVEGFR3 are mouse and human VEGFR3, respectively. (F) Uptake of HA-FITC in the differentiated ECs. Cells on the glass coverslips were treated with 25 μg HA-FITC for 2 hours. After fixation, the coverslips were visualized using a fluorescence microscope. The nuclei were counterstained with DAPI. Scale bar, 100 μm.

Expression of LYVE-1 was induced in VEGFR3-transfected ECs in response to sVEGF-C(WT). (A) HEK293 cells were transfected with VEGFR2 (top) or VEGFR3 (bottom) cDNAs. Transfected cells were treated with or without 400 ng/mL recombinant VEGF-C (rC), sVEGF-C(WT) (sC), and 40 ng/mL VEGF-A (A) for 20 minutes. Cell lysates were immunoprecipitated by anti-VEGFR2 (top panels) or anti-VEGFR3 (bottom panels) antibodies, followed by immunoblotting using antiphosphotyrosine (P-Tyr) antibody. Expression levels of VEGFR2 and VEGFR3 were confirmed by corresponding antibodies. (B) HEK293 cells were transfected with VEGFR2 and VEGFR3 cDNAs. Transfected cells were treated with or without sVEGF-C(WT) for 20 minutes. Cell lysates were immunoprecipitated by anti-VEGFR2 antibody, followed by immunoblotting using antiphosphotyrosine (P-Tyr, top) and anti-VEGFR2 (middle) antibodies. Immunoblotting for total cell lysates was performed using anti-VEGFR3 (bottom) antibody. (C) VEGFR2+ cells transfected with mock and VEGFR3 (long and short forms) cDNAs were cultured in SFO3 serum-free medium in the presence of supernatant (SFO3) of HEK293 cells transfected with mock (i) and VEGF-C cDNA (ii). After 2 days, cells were observed by phase-contrast microscopy (left panels) or doubly stained with anti–PECAM-1 (purple) and anti-SMA (brown) antibodies (right panels). Numbers of ECs and total cells were counted and shown as ECs/total (%). Scale bar, 100 μm. (D) Expression levels of VEGFR3 (long and short forms) in the differentiated ECs were analyzed by immunoblotting using anti-VEGFR3 antibody. Expression levels of PECAM-1 and α-tubulin were confirmed using specific antibodies (lower panels). M indicates mock; L, long; S, short. (E) RT-PCR analysis of lymphatic endothelial markers in the differentiated ECs described in panel C. mVEGFR3 and hVEGFR3 are mouse and human VEGFR3, respectively. (F) Uptake of HA-FITC in the differentiated ECs. Cells on the glass coverslips were treated with 25 μg HA-FITC for 2 hours. After fixation, the coverslips were visualized using a fluorescence microscope. The nuclei were counterstained with DAPI. Scale bar, 100 μm.

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