VEGF-C induces FLT-4 phosphorylation on HEL and THP-1 cells.

(A) Serum-starved leukemic cells were left untreated or stimulated with VEGF-C (100 ng/mL) for 10 minutes and then immunoprecipitated for tyrosine-phosphorylated proteins. Subsequent blots were probed with antibody against FLT-4. As a control, total FLT-4 levels are also shown (FLT-4 Western blot). (B) HEL cells placed in serum-free conditions were treated in the absence or presence of VEGF-C (100 ng/mL) for 10 minutes and then lysed with RIPA buffer. Lysates were immunoprecipitated for FLT-4, and blots were probed with anti-KDR antibodies. (C) HEL cells placed in serum-free conditions were treated in the absence or presence of AG1433 (2 μg/mL) for 1 hour and then treated with VEGF-C (100 ng/mL) or VEGF (20 ng/mL) for 10 minutes. Cells were then lysed with RIPA buffer and lysates immunoprecipitated for tyrosine-phosphorylated proteins. Subsequent blots were probed with anti-KDR or anti–FLT-4 antibodies. (D) Six primary leukemias were left untreated or stimulated with VEGF-C (100 ng/mL) for 10 minutes, immunoprecipitated for FLT-4, and probed with an antibody for phosphorylated proteins. As shown, FLT-4 induced tyrosine phosphorylation in FLT-4⁺ samples (nos. 1, 3, 4, 5, and 6). Bar graph represents the densitometry quantification of phosphorylated FLT-4 and is representative of 3 independent experiments.