VegfAb secretion and binding. Full-length vegfAa121, vegfAa165, vegfAb171, and vegfAb210 tagged with a V5 epitope at the N-termini were transfected into COS7 cells. (A) Western blot with a V5 antibody showed that although all 4 proteins are detectable in cell lysates (B) only VegfAa121 and VegfAa165 were secreted. (C) Transfected full-length kdra and kdrb fused to a V5 epitope were detectable in cell lysates of COS7 cells. (D) Replacing the endogenous signal peptide on all isoforms with the Igκ signal peptide resulted in secretion of both VegfAa and VegfAb. HEK 293 cells were transfected individually with Igκ-HA-vegfAa165-Myc, Igκ-HA-vegfAb171-Myc, soluble kdra-V5, and soluble kdrb-V5. Transfected cells were grown for 4 days, and conditioned media were collected, mixed, and incubated overnight. Immunoprecipitation (IP) with a V5 antibody (RTKs), blotting with Myc antibody (VegfAs), demonstrates that soluble Kdra bound to both VegfAa165 and VegfAb171; however, Kdrb did not appear to bind VegfAb171 as well as VegfAa165 in this assay. (E) Igκ-HA-vegfA-V5 constructs and Kdra-V5/pcDNA3.1 or Kdrb-V5/pcDNA3.1 were cotransfected into COS7 cells blotted with antiphosphotyrosine antibody, indicating activation of both RTKs by both VegfAa and VegfAb or (F) anti-V5 antibody demonstrating expression of the Kdra or Kdrb proteins.
