Effects of VEGF, PMA and TNFá on Cellular Tie2 Activation.

<p>HUVEC were treated with VEGF A, PMA B or TNFα C for 0, 0.5 or 24 h to induce Tie1 and Tie2 ectodomain cleavage followed by 200 ng/ml Ang1 for 30 min. Cells were lysed and phosphorylated Tie2 detected by immunoblotting with anti-phospho-Tie2 (pTie2), blots were stripped and re-probed for Tie1, Tie2 and tubulin. Phosphorylated Tie2 in cells treated with VEGF, PMA or TNFα for 0, 0.5 or 24 h and activated with Ang1 was detected by immunoblotting in at least three independent experiments and quantitated by densitometric scanning. Data are means and SEM of phospho-Tie2 normalized to that in Ang1-activated control cells within each experiment. Asterisks indicates significant increase in Ang1-induced Tie2 phosphorylation (p<0.05, Student's ‘t’test). D Loss of Tie1 enhances Ang1-activation of Tie2. HUVEC were transfected with contraol siRNA (C) or siRNA targeting Tie1 (Tie1) as indicated. 24 h post-transfection cells were activated with Ang1 for 30 min before lysis and detection of phosphorylated Tie2 (pTie2), blots were stripped and re-probed for Tie1 and Tie2. The relative mobility of mass markers is shown in kDa.