sVEGFR-3 is expressed in corneal epithelium and antagonizes VEGF-C. (A) Reverse-transcriptase PCRs (RT-PCRs) with intron-tail–specific reverse primer and exon-exon junction forward primers showing sVEGFR-3 in mouse cornea. Membrane VEGFR-3 mRNA expression in sclera only. (B) Western blot of corneal and scleral lysate (n = 5) with anti–VEGFR-3 N-terminal antibody demonstrates sVEGFR-3 at 60 kDa in cornea and membrane VEGFR-3 at 170 kDa in sclera. (C) Western blot of corneal and scleral lysate (n = 5) with anti–VEGFR-3 C-terminal antibody demonstrates the expression of membrane VEGFR-3 at 170 kDa in sclera only (none in cornea). (D) Immunolocalization of sVEGFR-3 (brown) in human cornea via an intron-derived C-terminal tail, human sVEGFR-3 antibody (PA 4835; Thermo Scientific). Isotype-negative control rabbit IgG. (E) RT-PCR and western blot of mouse cornea shows VEGF-C mRNA and protein. (F) Western blot of corneal lysate (n = 5) with anti–VEGFR-3 N-terminal antibody, blotted with anti–VEGF-C antibody under reducing (1) and native conditions (2), reveals binding of sVEGFR-3 to VEGF-C. (G) Sandwich ELISA with anti–VEGF-C–coated antibodies in 96-well plates, followed by the addition of corneal lysate (n = 5, each group; corneas from 3 mice in each sample), and then anti–VEGF-C, anti–VEGFR-3 N-terminal, and anti–N-terminal VEGFR-2 antibodies. (H) Competitive ELISA with human recombinant VEGF-C coated on 96-well plates and equimolar human recombinant VEGFR-3 and VEGFR-2 with extracellular domains added, showing affinity and binding to VEGF-C (n = 5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; M, marker; mVEGFR-3, membrane VEGFR-3; WB, western blot.