Phospho-KDR (Y1059) Antibody
Description
Definition and Target Specificity
Phospho-KDR (Y1059) antibody is a polyclonal antibody that specifically recognizes KDR phosphorylated at tyrosine residue 1059 (Y1059). This residue is part of the activation loop of KDR (also known as VEGFR2 or FLK-1), a receptor tyrosine kinase essential for angiogenesis, vascular permeability, and endothelial cell survival .
Key features:
-
Epitope: Phosphorylated tyrosine 1059 in the activation loop of human and mouse KDR .
-
Cross-reactivity: Predicted to react with bovine, canine, chicken, sheep, and zebrafish due to 100% sequence homology in the target region .
Applications and Validation
This antibody is validated for use in multiple experimental workflows:
Additional applications reported in literature include immunohistochemistry (IHC) and studies of VEGF-driven signaling pathways .
Role in VEGF Signaling
-
Phosphorylation at Y1059 is critical for KDR activation, enabling downstream signaling via PLCγ, MAPK, and AKT pathways .
-
In zebrafish, phospho-KDR (Y1059) antibody was used to demonstrate enhanced angioblast migration through VEGF sensitivity modulation .
Subcellular Localization
-
Studies using similar antibodies (e.g., anti-Y996) revealed phosphorylated KDR in cytoplasmic, perinuclear, and nuclear compartments of neoplastic cells, suggesting non-canonical signaling roles .
Supplier and Availability
-
Merck Millipore (ABS553): Sold as 200 µL affinity-purified antibody ($319 for 100 tests) .
-
Other vendors: Biorbyt and Fine Biotech offer alternatives, though with fewer validations .
Limitations and Considerations
Product Specs
Target Background
- Research suggests that miR-203a inhibits hepatocellular carcinoma cell invasion, metastasis, and angiogenesis by negatively targeting HOXD3 and suppressing cell signaling through the VEGFR pathway. PMID: 29402992
- Studies indicate that sFlt-1 up-regulation by VEGF may be mediated by the VEGF/Flt-1 and/or VEGF/KDR signaling pathways. PMID: 29497919
- miR424 might target VEGFR2 and inhibit Hemangioma-derived endothelial cell growth. PMID: 30132564
- VEGFR2 is regulated by deSUMOylation during pathological angiogenesis. PMID: 30120232
- A study demonstrated that decreasing the ratio of glutathione to oxidized glutathione with diamide leads to enhanced protein S-glutathionylation, increased reactive oxygen species (ROS) production, and enhanced VEGFR2 activation. PMID: 30096614
- Research confirmed the prognostic effect of EGFR and VEGFR2 for recurrent disease and survival rates in patients with epithelial ovarian cancer. PMID: 30066848
- No association was found between investigated VEGFR-2 gene polymorphisms and infantile hemangioma prognosis. PMID: 29984822
- Findings suggest functional interactions among ATX, VEGFR-2, and VEGFR-3 in modulating hemovascular and lymphovascular cell activation during vascular development. PMID: 30456868
- miR-195 suppresses cell proliferation of ovarian cancer cells through regulation of VEGFR2 and AKT signaling pathways. PMID: 29845300
- Thioredoxin-interacting protein (TXNIP) is highly induced in retinal vascular endothelial cells under diabetic conditions. Data, including from studies using knockout mice, suggest that TXNIP in retinal vascular endothelial cells plays a role in diabetic retinal angiogenesis via VEGF/VEGFR2 and Akt/mTOR signaling. PMID: 29203232
- Inhibition of FPR1 and/or NADPH oxidase functions prevents VEGFR2 transactivation and the triggering of the downstream signaling cascades. PMID: 29743977
- VEGFA activates VEGFR1 homodimers and AKT, leading to a cytoprotective response, while abluminal VEGFA induces vascular leakage via VEGFR2 homodimers and p38. PMID: 29734754
- An association of rs519664[T] in TTC39B on 9p22 with endometriosis has been reported. PMID: 27453397
- VEGF, VEGFR2, and GSTM1 polymorphisms have been investigated in the outcome of multiple myeloma patients treated with thalidomide-based regimens. PMID: 28665417
- In vitro studies showed that JFD-WS effectively inhibited HUVEC proliferation, migration, tube formation, and VEGFR2 phosphorylation. Additionally, JFD-WS inhibited blood vessel formation in the chick chorioallantoic membrane. While inhibiting xenograft tumor growth in experimental mice, JFD-WS decreased plasma MUC1 levels. PMID: 29436685
- The effects of Platelet-rich plasma on vascular endothelial growth factor receptor-2 (VEGFR2) and CD34 expression were evaluated using real-time PCR, flow cytometry, western blot, immunocytochemistry, and pathological study, in both human umbilical endothelial cell culture and rat skin. PMID: 28948378
- Metformin's dual effect in hyperglycemia-chemical hypoxia is mediated by a direct effect on VEGFR1/R2 leading to activation of cell migration through MMP16 and ROCK1 upregulation, and inhibition of apoptosis by an increase in phospho-ERK1/2 and FABP4, components of VEGF signaling cascades. PMID: 29351188
- Single nucleotide polymorphism of VEGFR2 is associated with relapse in gastroenteropancreatic neuroendocrine neoplasms. PMID: 29787601
- Data showed that ampelopsin inhibited angiogenesis with no cytotoxicity by suppressing both VEGFR2 signaling and HIF-1alpha expression. These results suggest that Hovenia dulcis Thunb. and its active compound ampelopsin exhibit potent antiangiogenic activities and could be valuable for the prevention and treatment of angiogenesis-related diseases including cancer. PMID: 29039561
- Authors demonstrated that when VEGFR2 was inhibited, NRP-1 appeared to regulate RAD51 expression through the VEGFR2-independent ABL-1 pathway, consequently regulating radiation sensitivity. Additionally, the combined inhibition of VEGFR2 and NRP-1 appears to sensitize cancer cells to radiation. PMID: 29777301
- Depletion of FGD5 in microvascular cells inhibited their migration towards a stable VEGFA gradient. Furthermore, depletion of FGD5 resulted in accelerated VEGFR2 degradation, which was reverted by lactacystin-mediated proteasomal inhibition. These findings suggest a mechanism whereby FGD5 sustains VEGFA signaling and endothelial cell chemotaxis via inhibition of proteasome-dependent VEGFR2 degradation. PMID: 28927665
- ATG5 and phospho-KDR expression was strongly associated with the density of vasculogenic mimicry in tumors and poor clinical outcome. PMID: 28812437
- Increased expression of VEGFR2 correlated with differentiation. PMID: 28854900
- DDA exhibits anti-angiogenic properties through suppressing VEGF-A and VEGFR2 signaling. PMID: 27517319
- RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF. PMID: 28271280
- Anlotinib occupied the ATP-binding pocket of VEGFR2 tyrosine kinase. PMID: 29446853
- The difference between the pro- (VEGF165a) and antiangiogenic (VEGF165b) VEGF isoforms and its soluble receptors for severity of diabetic retinopathy has been reported. PMID: 28680264
- Anlotinib inhibits the activation of VEGFR2, PDGFRbeta, and FGFR1 as well as their common downstream ERK signaling. PMID: 29454091
- Upregulation of sVEGFR-1 with concomitant decline of PECAM-1 and sVEGFR-2 levels in preeclampsia compared to normotensive pregnancies, irrespective of the HIV status, has been observed. PMID: 28609170
- By inhibiting the phosphorylation of VEGFR2, the P18 peptide (functional fragment of pigment epithelial-derived factor (PEDF) modulates signaling transduction between VEGF/VEGFR2 and suppresses activation of the PI3K/Akt cascades, leading to an increase in mitochondrial-mediated apoptosis and anti-angiogenic activity. PMID: 28627623
- VEGF increases arginine transport via modulation of CAT-1 in endothelial cells. This effect is exclusively dependent on KDR rather than Flt-1. PMID: 28478454
- This study shows that cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis. PMID: 27589831
- Eriocalyxin B inhibited VEGF-induced angiogenesis in HUVECs by suppressing VEGFR-2 signaling. PMID: 27756875
- The KDR fragment with domain 4 induced phosphorylation of VEGFR-2, as well as phosphorylation of downstream receptor kinases in HUVECs and VEGFR-2-positive breast cancer cells. PMID: 28303365
- Gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. PMID: 27713170
- Specificity protein 1 (Sp1) orchestrates the transcription of both VEGF and VEGFR2; hence, Sp1 could act as a therapeutic target. Research demonstrates that CF3DODA-Me induced apoptosis, degraded Sp1, inhibited the expression of multiple drivers of the blebbishield emergency program such as VEGFR2, p70S6K, and N-Myc through activation of caspase-3, inhibited reactive oxygen species, and inhibited K-Ras activation to abolis PMID: 28283889
- Icrucumab and ramucirumab are recombinant human IgG1 monoclonal antibodies that bind vascular endothelial growth factor (VEGF) receptors 1 and 2 (VEGFR-1 and -2), respectively. VEGFR-1 activation on endothelial and tumor cell surfaces increases tumor vascularization and growth and supports tumor growth via multiple mechanisms, including contributions to angiogenesis and direct promotion of cancer cell proliferation. PMID: 28220020
- The interplay among the ETS transcription factor ETV2, vascular endothelial growth factor, and its receptor VEGFR2/FLK1 is essential for hematopoietic and vascular development. Emerging studies support the role of these three factors and possible interplay in hematopoietic and vascular regeneration. PMID: 28026128
- DOT1L cooperates with transcription factor ETS-1 to stimulate the expression of VEGFR2, thereby activating ERK1/2 and AKT signaling pathways and promoting angiogenesis. PMID: 27626484
- This study provides new insights into the mechanism of VEGFR2 dimerization and activation. PMID: 28847506
- Cases with high MDSC infiltration, which was inversely correlated with intratumoral CD8(+) T-cell infiltration, exhibited shorter overall survival. In a mouse model, intratumoral MDSCs expressed both VEGFR1 and VEGFR2. VEGF expression in ovarian cancer induced MDSCs, inhibited local immunity, and contributed to poor prognosis. PMID: 27401249
- Results illustrated that CDK5-mediated KDR phosphorylation controls prolactin pituitary adenoma progression, and KDR pSer-229 serves as a potential prognostic biomarker for both noninvasive and invasive pituitary adenomas. PMID: 27438154
- Data indicate that simultaneous targeting of molecules that control distinct phases of angiogenesis, such as ALK1 and VEGFR, is a valid strategy for treatment of metastatic renal cell carcinoma (mRCC). PMID: 27248821
Customer Reviews
Applications : Phosphorylation assay
Sample type: cell
Review: The phosphorylation of VEGFR1 and VEGFR2 was examined using immunoblotting with rabbit polyclonal anti-phospho-VEGFR1 antibody and rabbit polyclonal anti-phospho-VEGFR2 antibody, respectively.
Q&A
What is Phospho-KDR (Y1059) Antibody and what does it detect?
Phospho-KDR (Y1059) antibody specifically detects endogenous levels of VEGFR-2 proteins only when phosphorylated at tyrosine 1059 . These antibodies are typically produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding tyrosine 1059 of human VEGFR-2 protein . They are available as polyclonal antibodies purified from rabbit antiserum by affinity chromatography using epitope-specific immunogens .
What is the significance of Y1059 in KDR/VEGFR-2 structure and function?
Y1059 is located in the tyrosine kinase catalytic domain (TKD2) of VEGFR-2 and plays a crucial role in kinase activity . This site, along with Y1054, is positioned in the activation loop (A-loop) of TKD2. When phosphorylated, these residues significantly increase the kinase activity of VEGFR-2 . The Y1059 residue is part of a critical regulatory mechanism for VEGFR-2 function in endothelial cells following VEGF stimulation.
How does the KDR/VEGFR-2 structure relate to its phosphorylation sites?
The VEGFR-2 structure can be divided into several functional domains with specific phosphorylation sites:
| Domain | Location | Key Phosphorylation Sites | Function |
|---|---|---|---|
| TKD1 | 834-930aa | - | ATP binding domain with hydrophobic pocket |
| KID | 931-998aa | Y951 | Kinase insert domain important for signaling |
| TKD2 | 999-1162aa | Y1054, Y1059 | Phosphotransferase domain with catalytic and activation loops |
| CTD | 1163-1356aa | Y1175, Y1214 | Critical for VEGFR-2 activation and downstream signaling |
The Y1059 site specifically influences VEGFR-2 kinase activity and is essential for proper receptor function .
What are the recommended experimental applications for Phospho-KDR (Y1059) antibodies?
Phospho-KDR (Y1059) antibodies are primarily used in Western Blotting (WB) with recommended dilutions of approximately 1:500-1:2000 . Some antibodies can also be used in ELISA applications with dilutions of around 1:5000 . The optimal dilutions should be determined by the researcher based on their specific experimental conditions . For Western Blot applications, these antibodies can detect proteins at approximately 230 kDa, corresponding to the phosphorylated form of VEGFR-2 .
How should I design experiments to study VEGF-induced KDR phosphorylation at Y1059?
For studying VEGF-induced KDR phosphorylation, a standard experimental design includes:
-
Culture endothelial cells (such as HUVECs) in appropriate media
-
Serum-starve cells overnight prior to stimulation
-
Stimulate cells with VEGF (typically 25 ng/mL VEGF165) for short periods (2-5 minutes)
-
Rapidly terminate the stimulation using ice-cold lysis buffer containing protease and phosphatase inhibitors
-
Perform cell lysis on ice (30-60 minutes) followed by centrifugation
-
Collect the protein-containing supernatant for analysis
-
Estimate protein concentration using standard methods (e.g., DC Protein Assay)
-
Detect phosphorylated KDR using Western blot with phospho-specific antibodies
This methodology allows for reliable detection of transient phosphorylation events at Y1059 following VEGF stimulation.
What controls should I include when using Phospho-KDR (Y1059) antibodies?
When using Phospho-KDR (Y1059) antibodies, include the following controls:
-
Positive control: VEGF-stimulated endothelial cells known to express phosphorylated VEGFR-2
-
Negative control: Unstimulated cells or cells treated with VEGFR-2 kinase inhibitors such as SU5416
-
Total KDR antibody: To normalize phospho-specific signals against total receptor expression
-
Loading control: Detection of housekeeping proteins to ensure equal loading across samples
-
Phosphatase-treated samples: To confirm specificity for phosphorylated epitopes
These controls help validate antibody specificity and experimental results.
How does Y1059 phosphorylation compare with other KDR phosphorylation sites?
Different phosphorylation sites on KDR/VEGFR-2 serve distinct signaling functions:
| Phosphorylation Site | Domain | Function | Comparison to Y1059 |
|---|---|---|---|
| Y1059 | TKD2 | Essential for kinase activity | Directly regulates enzymatic activity |
| Y951 | KID | Binds TSAd, regulates migration, survival, and permeability | More involved in downstream pathway activation than kinase regulation |
| Y1054 | TKD2 | Partners with Y1059 in kinase activation | Functions cooperatively with Y1059 |
| Y1175 | CTD | Binds PLCγ, p85 (PI3K), Shb; critical for proliferation | Key for PLCγ/PKC/MAPK pathway; mutated form is embryonic lethal |
| Y1214 | CTD | Involved in downstream signaling | Non-essential for development as Y1212F mutation is viable |
While Y1175 (Y1173 in mouse Flk-1) is essential for embryonic development, with Y1173F mutant mice dying at E8.5-E9.5 similar to Flk-1 null mice, Y1212F mutant mice are viable and fertile . Y1059, along with Y1054, serves a more fundamental role in regulating the catalytic activity of the receptor rather than directly recruiting specific signaling molecules.
What is the role of Y1059 phosphorylation in pathological conditions?
Phosphorylated KDR (pKDR) at Y1059 has been detected in various pathological conditions, particularly in cancer. In renal cell carcinomas (RCCs), pKDR has been found in the cell membrane, cytoplasm, and nuclei of both tumor cells and endothelial cells . This presence of pKDR in a wide variety of renal tumors suggests that anti-VEGF therapy might have direct effects on tumor cells beyond just targeting angiogenesis .
What methodological approaches can be used to study the functional consequences of Y1059 phosphorylation?
Several methodological approaches can be employed to study Y1059 phosphorylation:
-
Site-directed mutagenesis: Creating Y1059F mutants to study loss of phosphorylation at this site
-
Knock-in mouse models: Similar to studies done with Y1173F and Y1212F mutations in Flk-1
-
Phospho-specific antibodies: Using antibodies like Phospho-KDR (Y1059) for detection in various assays
-
Mass spectrometry: For unbiased identification of phosphorylation sites and quantification
-
Kinase inhibitors: Using specific inhibitors to modulate phosphorylation events
-
Phosphatase assays: To study the dynamics of phosphorylation/dephosphorylation
-
Intracellular trafficking studies: Using confocal microscopy to track receptor localization after phosphorylation
These approaches provide complementary data on the biological significance of Y1059 phosphorylation.
Why might I see weak or no signal when using Phospho-KDR (Y1059) antibodies?
Several factors could contribute to weak or absent signals:
-
Insufficient stimulation: VEGF stimulation might be too brief or at too low a concentration
-
Rapid dephosphorylation: Phosphatase activity may remove phosphates before detection
-
Protein degradation: Improper sample handling can lead to protein degradation
-
Antibody specificity: The antibody may not recognize the specific phosphorylated form in your experimental system
-
Species differences: Confirm the antibody recognizes your species of interest (human, mouse, rat)
-
Detection method sensitivity: Western blotting may require optimization of detection reagents
To troubleshoot, ensure proper VEGF stimulation (25 ng/mL for 2-5 minutes), use fresh phosphatase inhibitors in lysis buffers, and optimize antibody concentrations.
How can I differentiate between phosphorylation at Y1059 and other sites?
To differentiate between phosphorylation at different sites:
-
Use site-specific phospho-antibodies targeting distinct epitopes
-
Perform parallel experiments with antibodies against different phosphorylation sites
-
Use phosphopeptide competition assays to confirm specificity
-
Compare results with Y1059F mutant constructs where this site cannot be phosphorylated
-
Consider using mass spectrometry for unambiguous site identification
-
Combine with kinase inhibition studies that differentially affect specific phosphorylation sites
This multi-faceted approach helps disambiguate phosphorylation events at different tyrosine residues.
What are the emerging research directions regarding Y1059 phosphorylation?
Emerging research directions include:
-
Therapeutic targeting: Developing compounds that specifically inhibit phosphorylation at Y1059 or its downstream effects
-
Cross-talk with other receptors: Investigating how Y1059 phosphorylation is affected by or influences other receptor systems
-
Tumor-specific signaling: Understanding how Y1059 phosphorylation may differ in tumor versus normal endothelial cells
-
Biomarker potential: Evaluating whether Y1059 phosphorylation status could serve as a biomarker for disease progression or treatment response
-
Single-cell analysis: Examining heterogeneity in Y1059 phosphorylation at the single-cell level
-
Structural biology: Resolving how Y1059 phosphorylation precisely alters VEGFR-2 conformation and activity
These directions represent important frontiers in understanding the role of Y1059 in normal physiology and disease.
How do Y1059-directed antibodies compare with other tools for studying VEGFR-2 activation?
Each approach offers unique advantages and limitations, making them complementary in a comprehensive research program.
