Sunitinib blocked activation of VEGFR-2 and VEGFR-3 and their downstream molecules in human dermal LECs. LECs were cultured to confluence, and the medium was replaced with a serum-free medium. After overnight incubation, the cells were pretreated with sunitinib, anti-VEGFR2 antibody, or DMSO (controls), before being stimulated with 200 ng/ml VEGF-C (a) or VEGF-D (b). Lysates were immunoprecipitated with VEGFR-3-specific antibody for phospho-VEGFR-3 detection (c, d). Other phosphoproteins were detected with their respective specific antibodies. All samples were separated by 4% to 20% gradient SDS-PAGE gel. The proteins were blotted onto a PVDF membrane and detected by Western blotting. Tyrosine phosphorylation of VEGFR-2 and VEGFR-3 was noted in the cells treated with VEGF-C or VEGF-D. Sunitinib blocked phosphorylation of both VEGFR-3 and VEGFR-2, whereas anti-VEGFR-2 antibody inhibited phosphorylation of only VEGFR-2. Increased phosphorylation of ERK1/2 and Akt was observed in the LECs stimulated with the VEGFs; however, this was attenuated in the cells treated with sunitinib. DMSO, dimethyl sulfoxide; LEC, lymphatic endothelial cell; MW, molecular weight marker; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.