Phosphorylation of VEGF receptors and its downstream signals by PTK7. (A) Expression of PTK7 24 hours after transduction of siRNA silencing PTK7 (siPTK7), nonspecific random siRNA (siNeg), or lipofectamine only (control; **P < .01). (B) Phosphorylation of Flt-1 and KDR subsequent to PTK7-silencing siRNA transduction was determined after treatment with 20 ng/mL VEGF-A. When cells were > 90% confluent, transduction with PTK7-silencing or control siRNA was performed. Twenty-four hours later, cells were detached, resuspended, and reattached to wells of a 6-well plate. Twenty-four hours after stabilization, cells were treated with 20 ng/mL VEGF-A and collected for immunoblotting. Immunoblotting was performed using 10% (wt/vol) SDS-PAGE gels, followed by incubation with primary antibodies to each of phosphorylated Flt-1 (Y1213; 1:1000) and phospho-KDR (Y1054; 1:800). Bands were detected by chemiluminescence (**P < .01). The time courses of FAK (C) and Akt (D) phosphorylation after PTK7 silencing and VEGF-A treatment were determined by immunoblotting.