phospho-Tie-2 (Y992) Antibody

R&D Systems | Catalog # AF2720

R&D Systems
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Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human, Mouse

Cited:

Human, Mouse, Rat

Applications

Validated:

Immunohistochemistry, Western Blot, Intracellular Staining by Flow Cytometry, CyTOF-ready

Cited:

Immunohistochemistry, Western Blot, Immunocytochemistry, FACS

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG
View all Tie-2 Primary Antibodies »

Product Specifications

Immunogen

Phosphopeptide containing human Tie-2 Y992 site

Specificity

Detects human and mouse Tie-2 when phosphorylated at Y992 in Western blots.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for phospho-Tie-2 (Y992) Antibody

Detection of Mouse Phospho-Tie-2 (Y992) antibody by Western Blot.

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.
Phospho-Tie-2 (Y992) antibody in Human Kidney Tissue by Immunohistochemistry (IHC-P).

Phospho-Tie-2 (Y992) in Human Kidney Tissue.

Tie-2 was detected in immersion fixed paraffin-embedded sections of human kidney tissue using Rabbit Anti-Human/Mouse Phospho-Tie-2 (Y992) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2720) at 1 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (brown; Catalog # VC003) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in convoluted tubules. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.
Detection of Tie‑2 antibody in HUVEC Human Cells antibody 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 Tie-2 by Western Blot

Detection of Mouse Tie-2 by Western Blot

Pericyte-derived Angpt1 controls alveologenesis. a RT-qPCR analysis of Angpt1 and Tie2/Tek expression in freshly sorted lung GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. Data represents mean ± s.e.m. (n = 4 mice). b High magnification images of P10 Angpt1GFP lungs stained for GFP (green), PDGFR beta (red), and PDGFR alpha (blue). Arrows indicate GFP and PDGFR beta double positive pericytes. Scale bar, 15 µm. c RT-qPCR analysis of Angpt1 expression in freshly sorted PDGFR beta + cells from P7 Yap1,Wwtr1iPCKO and control lungs. Data represents mean ± s.e.m. (n = 4 mice, two-tailed unpaired t-test). dAngpt1 expression in cultured Verteporfin (VP)-treated (48 h) and control pericytes. Data represents mean ± s.e.m. (n = 4, Welch’s t-test). e Expression of the indicated transcripts in freshly sorted CD31+ cells from P7 Yap1,Wwtr1iPCKO and control lungs. Data represents mean ± s.e.m. (n = 4 mice, NS not significant, two-tailed unpaired t-test). f–h Western blot analysis of Angpt1 protein (f; n = 2 controls and 4 mutant mice) and of total and phospho-Tie2 (pTie2) in P12 Yap1,Wwtr1iPCKO and control total lung lysates (g, n = 3 controls and 5 mutants). Molecular weight marker (kDa) is indicated. Relative quantification of signals is shown in h. Two-tailed unpaired t-test. i Scheme showing the time points of tamoxifen administration and analysis for Angpt1iPCKO mice. j, k 3D reconstruction confocal images of P12 Angpt1iPCKO and littermate control lungs stained for AQP5 (green), PDGFR beta (red), and PECAM1 (blue). Panels in k show higher magnification of PECAM1 staining. Scale bar, 50 µm (j) and 30 µm (k). l Quantitation of airspace volume in P12 Angpt1iPCKO and littermate control lung sections with 3D reconstruction surface images. Data represents mean ± s.e.m. (n = 4 mice; p < 0.0001, two-tailed unpaired t-test). m 3D reconstruction confocal images of P12 Angpt1iPCKO and littermate control lungs stained for alpha SMA (red) and tropoelastin (blue). Scale bar, 50 µm. n Quantit
Detection of Phospho-Tie-2 (Y992) by Immunohistochemistry

Detection of Phospho-Tie-2 (Y992) by Immunohistochemistry

Tie2 activation reduces the level of phosphorylated VEGFR2 in GBM vessels.a Increase in phospho-Tie2 in GBM vessels by 4E2 treatment, with quantification (n = 5 per group). b Decrease in phospho-VEGFR2 in GBM vessels by 4E2 or DC101 treatment, with quantification (n = 5 per group). c Decrease in phospho-Akt in GBM vessels by 4E2 or DC101 treatment, with quantification (n = 5 per group). Scale bars: 100 µm (a–c). ns not significant; ****, P < 0.0001 vs. IgG. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36828931), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-Tie-2 (Y992) by Western Blot

Detection of Phospho-Tie-2 (Y992) by Western Blot

ER beta signals could increase Angiopoietin-2 and up-regulate the Angiopoietin-2/Tie-2 axis.a Protein expressions of ANGPT-2 of RCC cells and ANGPT-2, Tie-2 and p-Tie-2 of HUVEC cells. Western blot assays were performed on A498 cells transfected with pWPI+pLKO, oeER beta + pLKO, pWPI+shANGPT-2, oeER beta + shANGPT-2 (left panels), and on 786-O cells transfected with pLKO+pWPI, shER beta + pWPI, pLKO+oeANGPT-2, shER beta + oeANGPT-2 (right panels). b–e Tube formation assays were performed using (b) A498 cells transfected with pWPI+pLKO, oeER beta + pLKO, pWPI+shANGPT-2, oeER beta + shANGPT-2, using (c) A498 cells transfected with pWPI+pLKO, oeER beta + pLKO, pWPI+shANGPT-2, oeER beta + shANGPT-2, and treated with1 μM Sunitinib, using (d) 786-O cells transfected with pLKO+pWPI, shER beta + pWPI, pLKO+oeANGPT-2, shER beta + oeANGPT-2 and (e) using 786-O cells transfected with pLKO+pWPI, shER beta + pWPI, pLKO+oeANGPT-2, shER beta + oeANGPT-2, and treated with1 μM sunitinib. For b–e, quantitation is at the right of the images. Data are presented as mean ± SD, *p < 0.05; **p < 0.01, compared with the controls, and Scale bar: 5 μm. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32409702), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-Tie-2 (Y992) by Western Blot

Detection of Phospho-Tie-2 (Y992) by Western Blot

AB‐Tie1‐39 treatment stabilizes lung vessels to restrict extravasation of disseminated tumor cellsAMice were pretreated with IgG or AB‐Tie1‐39. Thereafter, B16F10 cells were intravenously injected to initiate experimental metastasis independent of the primary tumor.BRepresentative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB‐Tie1‐39 = 8 mice). *P < 0.05 (two‐tailed Mann–Whitney U‐test).CECs were seeded on gelatin‐coated Transwell inserts. RFP‐labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***P < 0.001 (two‐tailed Mann–Whitney U‐test).DLLC tumor‐bearing mice were pretreated with IgG or AB‐Tie1‐39. On day 14, lung tissue lysates were immunoblotted with anti‐phospho‐TIE2, total‐TIE2, and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **P < 0.01 (two‐tailed Mann–Whitney U‐test).ELLC tumor‐bearing mice were pretreated with IgG or AB‐Tie1‐39. On day 14, lung ECs were FACS‐sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly regulated genes (red outline = AB‐Tie1‐39‐treated; black outline = IgG‐treated). On the right, top significantly regulated bio‐functions in AB‐Tie1‐39‐treated as compared to IgG‐treated mice in ingenuity pathway analysis.Source data are available online for this figure. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32302470), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-Tie-2 (Y992) by Immunohistochemistry

Detection of Phospho-Tie-2 (Y992) by Immunohistochemistry

Antibody-based Tie2 activation attenuates VEGFR2 phosphorylation via VE-PTP.a–c Immunoblots (a) showing VE-PTP-mediated VEGFR2 dephosphorylation by 4E2 and quantification of the phospho-VEGFR2 (b, n = 3 per group) and phospho-Tie2 (c, n = 3 per group) levels. AKB-9778, inhibitor of VE-PTP. d Quantification of VEGF-induced dextran leakage in HUVECs (n = 3 per group). e–g Immunostaining and quantification of phospho-VEGFR2 (e), phospho-Tie2 (f), and VE-cadherin (g) in GBM vessels cotreated with 4E2 and AKB-9778 (n = 5 per group). h Increased immunoprecipitation of VE-PTP with VEGFR2 but not Tie2 by 4E2. i, j Quantification of the normalized VEGFR2/VE-PTP (i, n = 3 per group) and normalized Tie2/VE-PTP (j, n = 3 per group) levels. k Schematic showing the regulatory mechanisms of tumor vascular normalization by 4E2. EC, endothelial cell; PC, pericyte; BM, basement membrane. Scale bars: 100 µm (e–g). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. Ctrl (c, h-i) or IgG (e-g). ns not significant; #, P < 0.05; ##, P < 0.01 vs. VEGF (b, d) or 4E2 (e–g). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36828931), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for phospho-Tie-2 (Y992) Antibody

Application
Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Immunohistochemistry

1-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human kidney tissue

Intracellular Staining by Flow Cytometry

0.25 µg/106 cells
Sample: HUVEC human umbilical vein endothelial cells treated with pervanadate, fixed with paraformaldehyde, and permeabilized with methanol

Western Blot

0.5 µg/mL
Sample: NIH‑3T3 mouse embryonic fibroblast cell line transfected with mouse Tie-2 and treated Recombinant Human Angiopoietin‑1 (Catalog # 923-AN)

Reviewed Applications

Read 3 reviews rated 3.7 using AF2720 in the following applications:

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Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

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Background: Tie-2

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.

Long Name

Tyrosine Kinase with Immunoglobulin and Epidermal Growth Factor Homology Domains 2

Alternate Names

CD202b, TEK, Tie2

Entrez Gene IDs

7010 (Human); 21687 (Mouse); 89804 (Rat); 396729 (Porcine); 403714 (Canine); 102122204 (Cynomolgus Monkey); 30747 (Zebrafish)

Gene Symbol

TEK

Additional Tie-2 Products

Product Documents for phospho-Tie-2 (Y992) Antibody

Certificate of Analysis

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Product Specific Notices for phospho-Tie-2 (Y992) Antibody

For research use only

Citations for phospho-Tie-2 (Y992) Antibody

Customer Reviews for phospho-Tie-2 (Y992) Antibody (3)

3.7 out of 5
3 Customer Ratings
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Showing  1 - 3 的 3 reviews Showing All
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  • Human/Mouse Phospho-Tie-2 (Y992) Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: HUVEC human umbilical vein endothelial cells
    Species: Human
    Verified Customer | Posted 01/06/2026
    After trying several pTIE2 Y992 antibodies I have realized that this antibody works on HUVEC and its variant cells. It would usually give some other bands, but the top upper band at around 160 kDa usually corresponds to pTIE2 on my HUVEC/L914F cells.
    phospho-Tie-2 (Y992) Antibody AF2720
  • Human/Mouse Phospho-Tie-2 (Y992) Antibody
    Name: Anonymous
    Application: Immunofluorescence in mouse brain tissue
    Sample Tested: Cerebellum tissue
    Species: Mouse
    Verified Customer | Posted 10/02/2020
    Immunofluorescence with phospho Tie2
    Postnatal mouse cerebellum at P14 was used to determine the levels of phospho-Tie2 in the blood vessels. The blocking buffer and the antibody buffers were prepared in 0.2% gelatin in PBST (In primary and secondary solutions were used 0.025% of Saponin was added). The sections were antigen retrieved by boiling in a microwave for 5 min using 10mM Sodium citrate (pH6).
    phospho-Tie-2 (Y992) Antibody AF2720
  • Human/Mouse Phospho-Tie-2 (Y992) Antibody
    Name: Mohammad Daud Khan
    Application: Western Blot
    Sample Tested: HASMC human aortic smooth muscle cells
    Species: Human
    Verified Customer | Posted 10/17/2018
    phospho-Tie-2 (Y992) Antibody AF2720

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