Phospho-Tie2 (Y992) assay principle

The Phospho-Tie2 (Tyr992) assay measures Tie2 when phosphorylated at Tyr992. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its ability to recognize the protein independently of its phosphorylation state, the second for its specific binding to the phosphorylated motif on the protein. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-Tie2 (Y992) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-Tie2 (Tyr992) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-Tie2 (Tyr992) one-plate assay protocol

Detection of Phosphorylated Tie2 (Tyr992) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Angiopoietin 1 induces Tie2 phosphorylation

200,000 Huvec cells were plated on a 96-well plate after 30 minutes of polyornithine treatment. Cells were incubated 24 hours at 37°C before specific stimulation. Concentrations of pervanadate (25µM and 12.5µM) were tested in presence or absence of Angiopoietin 1 at 800 ng/mL for 15 minutes. These solutions were prepared in a cell culture medium without serum. After treatment at 37°C, culture medium was removed and 50µL of complemented lysis buffer was added for 30 minutes under shaking. Lysates were then transfered into a 384-well plate for Phospho Tie2 detection.

In presence of pervanadate, Angiopoietin 1 induced an increase of Tie2 phosphorylation at Tyr992, highlighting the specific activation of the Tie2 pathway.

Tie2 Signaling Pathway

Tie-2 (TEK receptor tyrosine kinase) protein induces angiogenesis and supports endothelial cell survival, upon binding of an endothelial growth factor, angiopoietin-1 (Angpt1), to the receptor. In presence of Angiopoietin 1, receptors form homodimers followed by the autophosphorylation of the C-terminal tails at Tyrosine 992 and activation of the downstream signaling pathways including MEK/ERK, IKKb/NFkB and AKT. The overall activation results in the maintenance of vascular stability, cellular proliferation, inflammation and cell survival. The downregulation is induced by two mechanisms: deactivation of active Tie2 by vascular endothelial protein tyrosine phosphatase (VE-PTP) and binding of the non-activating ligand angiopoietin-2 (Angpt2).