Intracellular Staining by Flow Cytometry
Detection of Tie‑2 in HUVEC Human Cells by Flow Cytometry. HUVEC human umbilical vein endothelial cells were unstimulated (light orange filled histogram) or treated with 100 μM pervanadate for 15 minutes (dark orange filled histogram), then stained with Human/Mouse Phospho-|
Tie‑2 (Y992) Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF2720) or control antibody (Catalog # AB-105-C, open histogram), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.
Detection of Mouse Phospho-Tie‑2 (Y992) by Western Blot. Western blot shows lysates of NIH‑3T3 mouse embryonic fibroblast cell line transfected with mouse Tie-2 and untreated (-) or treated (+) with |
600 ng/mL Recombinant Human Angiopoietin‑1 (Catalog # 923-AN) for 5 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human/Mouse Phospho-Tie‑2 (Y992) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2720) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-Tie‑2 (Y992) at approximately 150 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Tie-2 (also known as TEK) is an angiogenic receptor tyrosine kinase required for the later stage of blood vessel maturation. Ligand binding induces receptor dimerization and autophosphorylation on multiple tyrosine residues. Y992 is located on the putative activation loop of Tie-2 and is a major autophosphorylation site.
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