Figure 7. VE-PTP counterbalances Ang1 activation of Tie-2 and regulates Tie-1 tyrosine phosphorylation dependent on Tie-2. (A) bEnd.5 cells were either transfected with control siRNAs or with siRNAs directed against VE-PTP. 24 h later, cells were stimulated either with 600 ng/ml Ang1 or 600 ng/ml Ang2 for 10 min, or unstimulated (unstim). Immunoprecipitates of Tie-2 (top two panels) or cell lysates (bottom two panels) were immunoblotted for the indicated antigens. (B) HUVECs were either transfected with control siRNAs or with siRNAs directed against hVE-PTP. 24 h later, siRNA-transfected cells (left two lanes) and untransfected cells, stimulated either with 600 ng/ml Ang1 for 10 min or unstimulated (right two lanes), were subjected to immunoprecipitations for Tie-2, followed by immunoblotting with antibodies against the phosphorylated tyrosine 992 in the active loop of the kinase (pTyr 992) and Tie-2. Aliquots of cell lysates were immunoblotted directly for hVE-PTP and Tie-2 (bottom two panels). White line indicates that intervening lanes have been spliced out. (C) bEnd.5 cells were treated either with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for 1 h, followed by either Ang1 stimulation for 10 min (Ang1) or no stimulation (unstim). Phosphorylation of tyrosine 992 was analyzed as described for B. Quantified signal intensities are indicated. (D) bEnd.5 cells were stimulated with 200 ng/ml COMP-Ang1 for 10 min or left untreated. VE-PTP immunoprecipitates (top three panels) and cell lysates (bottom two panels) were analyzed by immunoblotting for Tie-2, VE-cadherin, and VE-PTP as indicated on the right. (E) Embryonic endothelioma cells established either from wild-type (+/+) or Tie-2–deficient (−/−) embryos were antibody pretreated as indicated and subjected to immunoprecipitations with antibodies against Tie-1. Immunocomplexes were analyzed by immunoblotting with antibodies against phosphotyrosine or Tie-1 (as indicated). Aliquots of cell lysates were immunoblotted directly for Tie-1 and Tie-2 (bottom two panels).
Figure 7.

VE-PTP counterbalances Ang1 activation of Tie-2 and regulates Tie-1 tyrosine phosphorylation dependent on Tie-2. (A) bEnd.5 cells were either transfected with control siRNAs or with siRNAs directed against VE-PTP. 24 h later, cells were stimulated either with 600 ng/ml Ang1 or 600 ng/ml Ang2 for 10 min, or unstimulated (unstim). Immunoprecipitates of Tie-2 (top two panels) or cell lysates (bottom two panels) were immunoblotted for the indicated antigens. (B) HUVECs were either transfected with control siRNAs or with siRNAs directed against hVE-PTP. 24 h later, siRNA-transfected cells (left two lanes) and untransfected cells, stimulated either with 600 ng/ml Ang1 for 10 min or unstimulated (right two lanes), were subjected to immunoprecipitations for Tie-2, followed by immunoblotting with antibodies against the phosphorylated tyrosine 992 in the active loop of the kinase (pTyr 992) and Tie-2. Aliquots of cell lysates were immunoblotted directly for hVE-PTP and Tie-2 (bottom two panels). White line indicates that intervening lanes have been spliced out. (C) bEnd.5 cells were treated either with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for 1 h, followed by either Ang1 stimulation for 10 min (Ang1) or no stimulation (unstim). Phosphorylation of tyrosine 992 was analyzed as described for B. Quantified signal intensities are indicated. (D) bEnd.5 cells were stimulated with 200 ng/ml COMP-Ang1 for 10 min or left untreated. VE-PTP immunoprecipitates (top three panels) and cell lysates (bottom two panels) were analyzed by immunoblotting for Tie-2, VE-cadherin, and VE-PTP as indicated on the right. (E) Embryonic endothelioma cells established either from wild-type (+/+) or Tie-2–deficient (−/−) embryos were antibody pretreated as indicated and subjected to immunoprecipitations with antibodies against Tie-1. Immunocomplexes were analyzed by immunoblotting with antibodies against phosphotyrosine or Tie-1 (as indicated). Aliquots of cell lysates were immunoblotted directly for Tie-1 and Tie-2 (bottom two panels).

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