Specificity of AFL4. (A) AFL-4 binding to VEGFR-1-Fc, VEGFR-2-Fc, or VEGFR-3-Fc was analyzed by ELISA. The absorbance at 450 nm is depicted. Each bar represents the mean ± SEM of triplicate assays. (B) Cell lysates from the 293 cell line transiently transfected with murine VEGFR-3 DNA (lane 2) or mock transfected (lane 1, M) were precipitated with AFL4 and immunoblotted with the same antibody. (IP, immunoprecipitation.) Two bands of 195- and 125-kd proteins were detected in lane 2 under reducing conditions. Through immunoblotting of the cell lysates from the murine EC line F2 with anti-VEGFR-3 mAb, 2 bands were detected in nonreducing conditions (NR), whereas only a 125-kd band was seen under reducing conditions (R) (lanes 3, 4). Arrows denote the positions of the unprocessed 195-kd form and the proteolytically processed 125-kd form of VEGFR-3. (C) Specificity of AFL4 in tissue sections. Sagittal sections were prepared from embryonic day 9.5 VEGFR-3^−/− or VEGFR-3^+/+ embryos and stained with AFL4, anti-PECAM-1 mAb, and anti-VEGFR-2 mAb. Arrows indicate blood vessels in the mesenchymal region around the neural tube (NT). Note that all PECAM-1⁺cells are also VEGFR-2⁺ and VEGFR-3⁺ at this stage of embryonic development.