sVEGFR-3 knockdown leads to growth of corneal lymphatic and blood vessels and expression and phosphorylation of membrane VEGFR-3. (A) Immunofluorescent staining and confocal imaging of cornea flat mounts (n = 10 each group) injected with 2 μg pshRNA–sVEGFR-3, pshRNA-CTR, pshRNA–sVEGFR-3 + anti–VEGF-C immunoglobulin, and IgG. pshRNA–sVEGFR-3 injection leads to lymphangiogenesis in 10 days, with vessels oriented to injection site. Anti–VEGF-C antibody administration 1 day before pshRNA-sVEGFR-3 controls lymphangiogenesis. (B-C) Corneal area of blood and lymphatic vessels 10 days after pshRNA–sVEGFR-3, pshRNA-CTR, pshRNA–sVEGFR-3 + anti–VEGF-C immunoglobulin, and IgG antibody injection. Anti–VEGF-C immunoglobulin injection 1 day prior to pshRNA–sVEGFR-3 shows 75% and 56% reduction of lymphatic and blood vessels (n = 10 each group). (D) sVEGFR-3 knockdown with pshRNA–sVEGFR-3 injection into cornea is associated with expression of membrane VEGFR-3 and downregulation of sVEGFR-3 as seen in western blot with anti–N-terminal VEGFR-3 antibody. (E) sVEGFR-3 knockdown with pshRNA–sVEGFR-3 (samples harvested on day 5 after injection) is associated with phosphorylation of membrane VEGFR-3 as demonstrated via immunoprecipitation with anti–N-terminal VEGFR-3 antibody followed by western blotting with anti-phosphotyrosine antibody. (F) Stripping and reblotting the membrane with anti–N-terminal end VEGFR-3 antibody confirms that sVEGFR-3 is present after control shRNA treatment but not after pshRNA–sVEGFR-3. *P < .01 by paired Student t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; WB, western blot.