Fig. 2. Inhibition of VEGFR-3 function by the mAb hF4-hF4-3C5. (A) Expression of chimeric VEGFR-3-cFms receptor by NIH-3T3 cells (clone 3.1.2, lines 2 and 4) or control NIH 3T3 cells (lanes 1 and 3) was determined by western blotting with rabbit antibodies to cFms (lanes 1 and 2) or to the extracellular domain of VEGFR-3 (lanes 3 and 4). Lysates in lanes 2 and 4 contain a single polypeptide that is recognized by the antibody to the C-terminus of cFms and an antibody to the extracellular domain of VEGFR-3. (B) Clone 3.1.2 cells were analyzed by FACS with either mAb hF4-3C5 or the control mAb 2F8. The plasma membrane expression of VEGFR-3-cFms is evident. (C) Stimulation of mitogenic response of the clone 3.1.2 cells with VEGF-C_NC results in dose-dependent [³H] thymidine uptake with an EC₅₀ value of 3.3 ng/ml. (D) Clone 3.1.2 cells were stimulated with 80 ng/ml VEGF-C_NC in the presence of variable amounts of mAb hF4-3C5 or the control antibody 2F8. The maximum inhibition of the response is seen between 10 and 15 nM hF4-3C5 with an IC₅₀ of 5 nM (750 ng/ml). mAb 2F8 had no effect on [³H] thymidine uptake.