Expression and ligand binding of recombinant alternative splice variants. (a) HEK293 cells were transiently transfected with the indicated cDNA constructs. Conditioned media of HEK293 cells were collected after 48 hours, separated on SDS-PAGE gels and probed with an anti-Myc antibody to detect the Myc-tagged alternative splice variants (ASV). Molecular weights (kDa) are indicated. (b) For VEGFR1-541, VEGFR2-712, PDGFRβ-336, Met-877 and CSF1R-306, conditioned media from untransfected (Control, dashed lines) or ASV-transfected (Specific, solid lines) HEK293 cells were applied to plates precoated with the receptor-specific ligands. Unbound ASV were detected using antibodies against the extracellular domains of the receptors. Purified Tie1-751(6His) was used for Ang-1 binding, as above. Kd, dissociation constant. (c) Solution binding of VEGF-D to VEGFR3-765-Myc. Binding was carried out by combining VEGF-D with conditioned medium from either VEGFR3-765-Myc-expressing cells (lanes 1 to 3) or untransfected cells (lane 4). Subsequent immunoprecipitation was performed using anti-VEGF-D antibody and detected using anti-Myc antibody. To confirm the specificity of interaction between VEGF-D and VEGFR3-765-Myc, binding was performed in the presence of fivefold molar excess of either recombinant human VEGFR3/Fc chimera (lane 2) or soluble recombinant human VEGFR1/Fc chimera (lane 3). Molecular weights (kDa) are indicated. CM, Conditioned medium; IP, Immunprecipitation; WB, Western blot.