AFL4 blocks VEGFR-3 function by inhibiting VEGF-C binding.

(A) Inhibition of binding of VEGFR-3 to VEGF-C by AFL4. Culture supernatant of 293 cells transfected with 5 myc-tagged VEGF-C DNA was incubated with various doses of AFL4 (•) or anti-VEGFR-2 mAb (○) and added to microtiter plates coated with mVEGFR-3-Fc. Binding was quantified by using the anti-myc mAb as a primary antibody, and absorbance at 450 nm was determined. Data indicate the background-corrected mean ± SEM from triplicate wells. (B) Tyrosine phosphorylation of VEGFR-3 in F2 cells was induced by VEGF-C CM in the presence of control IgG (lanes 1, 3) or AFL4 (lanes 2, 4). Total cell lysates were immunoprecipitated with anti-VEGFR-3 mAb and subjected to serial immunoblotting with anti-phosphotyrosine antibody (upper) and anti-VEGFR-3 mAb (lower). Arrows and arrowheads denote the positions of 195-kd and 125-kd forms of VEGFR-3. Relative density of bands against 125-kd bands of the control lanes (lane 1, upper and lower panels) were 195-kd/125-kd 3.7/1 (lane 1, upper), 0.9/0.2 (lane 2, upper), 0.78/1 (lane 1, lower), and 0.6/1.1 (lane 2, lower). Reduction ratios after correction by the amount of protein were 0.32 and 0.18 for 195-kd and 125-kd bands, respectively.