Detection of Tie2 Cell-Associated Endodomain.

<p>A Cells were incubated with PMA for the times indicated in hours before cell lysis and detection of cellular Tie1 endodomain by immunoblotting with an antibody recognizing Tie1 carboxy-terminus, as indicated. Blots were stripped and re-probed with an antibody to Tie2 intracellular domain, as indicated. The positions of molecular mass markers in kDa are shown on the left of the blots. B To confirm the 55 kDa Tie2 immunoreactive fragment is derived from Tie2 endothelial cells were transfected with control siRNA (Scr) or siRNA recognizing Tie2, as indicated. 24 h post-transfection cells were incubated with or without PMA for 24 h before lysis and detection of Tie2 endodomain by immunoblotting. C VEGF stimulates Tie2 endodomain accumulation at 24 h. Endothelial cells were incubated with VEGF for 0.5 or 24 h before cell lysis and detection of cellular Tie2 intracellular domain. Membranes were stripped and reprobed for Tie1 intracellular domain. D Effects of γ-secretase inhibition on Tie1 and Tie2 endodomains formed in response to PMA. HUVEC were incubated with or without the γ -secretase inhibitor in the absence and presence of PMA for 0.5 or 24 h as indicated. Tie1 endodomain was detected in whole cell lysates by immunoblotting with an antibody recognizing Tie1 carboxy-terminus and blots were stripped and re-probed with an antibody to Tie2 intracellular domain as indicated. Effects of γ-secretase inhibition on Tie1 and Tie2 endodomains formed in response to VEGF (E) and TNFα (F). HUVEC were incubated with or without the γ -secretase inhibitor in the absence and presence of VEGF or TNFα for 0.5 or 24 h as indicated. Tie1 endodomain was detected in whole cell lysates by immunoblotting with an antibody recognizing Tie1 carboxy-terminus and blots were stripped and re-probed with an antibody to Tie2 intracellular domain as indicated. G Tie1 endodomain interacts with full-length Tie2. HUVEC were incubated with VEGF or TNFα for 0.5 or 24 h as indicated. Cells were lysed and Tie2 immunoprecipitated. Tie1 endodomain co-immunoprecipitating with Tie2 was detected by immunoblotting with an antibody recognizing Tie1 carboxy-terminus and blots were stripped and re-probed with an antibody to Tie2. The relative mobility of mass markers is shown in kDa.