Phospho-FLT1 (Tyr1213) Antibody

What is Phospho-FLT1 (Tyr1213) and why is it significant in vascular biology research?

Phospho-FLT1 (Tyr1213) refers to the FLT1 protein (also known as VEGFR1) when phosphorylated at the tyrosine 1213 residue. This specific phosphorylation site is considered the main auto-phosphorylation site responsible for activation of intracellular signaling pathways downstream of VEGFR1. The significance of this phosphorylation lies in its role as a critical marker of receptor activation following ligand binding. Tyrosine 1213 phosphorylation occurs when VEGFR1 binds to its ligands, primarily VEGF-A and PlGF, triggering signal transduction cascades that regulate angiogenesis, vasculogenesis, and cell migration . For researchers studying vascular development, tumor angiogenesis, or inflammatory responses, detecting phosphorylation at this specific residue provides crucial information about the activation status of VEGFR1 signaling in their experimental systems.

What is the difference between FLT1 and VEGFR1 terminology?

FLT1 (Fms-like tyrosine kinase 1) and VEGFR1 (Vascular Endothelial Growth Factor Receptor 1) refer to the same protein. FLT1 is the official gene name, while VEGFR1 describes its functional role as a receptor for VEGF. The protein is also sometimes referred to as FLT, FRT, or vascular permeability factor receptor . This receptor belongs to the receptor tyrosine kinase (RTK) family and contains an extracellular domain with seven immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic tyrosine kinase domain . In scientific literature, both terms are used interchangeably, but VEGFR1 is often preferred when discussing the protein in the context of VEGF signaling pathways and angiogenesis research, while FLT1 may be more commonly used in genetic contexts or when referring specifically to the gene.

What validated applications can Phospho-FLT1 (Tyr1213) antibodies be used for?

Phospho-FLT1 (Tyr1213) antibodies have been validated for several experimental applications:

The antibody has been most extensively validated for Western blot applications, where it has been shown to detect a specific band for Phospho-VEGFR1/Flt-1 (Y1213) at approximately 130-140 kDa under reducing conditions . When selecting an application, researchers should consider the specific antibody formulation and follow the manufacturer's recommended protocols for optimal results.

How does phosphorylation at Tyr1213 relate to FLT1/VEGFR1 function?

Tyrosine 1213 is regarded as the main auto-phosphorylation site responsible for activation of intracellular pathways downstream of VEGFR1 . Unlike VEGFR2, VEGFR1 exhibits weak tyrosine kinase activity upon ligand binding, making the phosphorylation events particularly significant for understanding its signaling mechanisms.

The phosphorylation process occurs as follows:

• Binding of ligands (VEGF-A or PlGF) to VEGFR1

• Receptor dimerization

• Auto-phosphorylation at multiple tyrosine residues, including Tyr1213

• Activation of downstream pathways, including Erk1/2 phosphorylation

This phosphorylation is essential for VEGFR1's roles in angiogenesis, vasculogenesis, inflammatory responses, and monocyte migration. In pathological contexts like glioblastoma multiforme (GBM), VEGFR1 phosphorylation at Tyr1213 has been observed in highly VEGFR-1-expressing cell lines upon exposure to exogenous VEGF-A or PlGF, suggesting its importance in tumor-associated angiogenesis .

How do VEGF-A and PlGF differentially induce phosphorylation of FLT1 at Tyr1213?

Research has shown that:

• VEGF-A binds to both VEGFR1 and VEGFR2, while PlGF binds exclusively to VEGFR1

• In U87-derived cells over-expressing VEGFR-1, both ligands induced significant phosphorylation at Tyr1213

• Anti-PlGF antibodies only partially affected growth factor-induced VEGFR-1 auto-phosphorylation at Tyr1213, suggesting differential mechanisms

• The D16F7 monoclonal antibody prevents VEGFR-1 auto-phosphorylation in response to both ligands, indicating a common pathway that can be blocked

These differences are important for researchers designing experiments to study VEGFR1 activation, as the choice of stimulating ligand may influence the experimental outcomes and interpretation of results.

What downstream signaling pathways are activated following Tyr1213 phosphorylation?

Following phosphorylation at Tyr1213, VEGFR1 activates several downstream signaling cascades that regulate cellular responses. The primary pathway identified in the research literature is the MAPK/ERK pathway:

• Phosphorylation of VEGFR1 at Tyr1213 leads to downstream phosphorylation of Erk1/2 (extracellular signal-regulated kinases 1/2)

• This activation can be counteracted by treatment with specific antibodies like D16F7

• The activated ERK pathway contributes to cell migration, proliferation, and survival

Other potential downstream effects of Tyr1213 phosphorylation include:

• Activation of PI3K/Akt pathway components

• Regulation of inflammatory responses

• Promotion of cell motility and invasion

• Contribution to angiogenic processes

The specific downstream effects may vary depending on cell type and experimental conditions, making it important for researchers to validate the relevant pathways in their specific experimental systems.

How does phosphorylation at Tyr1213 compare with other phosphorylation sites on FLT1?

VEGFR1/FLT1 contains multiple tyrosine phosphorylation sites, each potentially contributing to different aspects of receptor function. While Tyr1213 is considered a main auto-phosphorylation site, other sites include Tyr1048, Tyr1169, Tyr1327, and Tyr1333.

Tyr1213 phosphorylation appears to be particularly important for VEGFR1 signaling in the context of tumor angiogenesis and progression. Research in glioblastoma models has shown that this phosphorylation site becomes active upon ligand binding and contributes to downstream signaling events . The relative importance of different phosphorylation sites may vary depending on cellular context and experimental conditions.

What are the best experimental controls when studying phosphorylation at Tyr1213?

When studying phosphorylation at Tyr1213 of VEGFR1/FLT1, implementing appropriate controls is crucial for experimental validity:

Positive Controls:

• Pervanadate treatment: Cell lines (such as HUVEC) treated with pervanadate (100 μM for 10 minutes) show enhanced tyrosine phosphorylation and can serve as positive controls

• VEGF-A or PlGF stimulation: Cells expressing VEGFR1 stimulated with VEGF-A or PlGF (typically 50 ng/ml) for short periods

Negative Controls:

• Unstimulated cells: Cells maintained in serum-free or low-serum conditions without growth factor stimulation

• Phosphatase treatment: Treating samples with phosphatases to remove phosphorylation

• Blocking peptide controls: Using the phosphopeptide immunogen to demonstrate antibody specificity

Specificity Controls:

• Comparison with total VEGFR1 antibody detection to determine the proportion of phosphorylated receptor

• Use of species- and isotype-matched control antibodies (e.g., mouse IgG1) in parallel experiments

• VEGFR1 knockdown or knockout cells to confirm signal specificity

These controls help ensure that detected signals are specific to phosphorylated Tyr1213 and not due to non-specific antibody binding or experimental artifacts.

What are the optimal sample preparation methods for detecting Phospho-FLT1 (Tyr1213)?

Proper sample preparation is critical for successful detection of Phospho-FLT1 (Tyr1213). The following protocol incorporates best practices from multiple sources:

• Cell Stimulation:

  • Starve cells in serum-free medium for 4-24 hours

  • Stimulate with VEGF-A or PlGF (50 ng/ml) for 5-15 minutes

  • Alternatively, treat with pervanadate (100 μM) for 10 minutes to maximize phosphorylation

• Lysis Procedure:

  • Rapidly place cells on ice and wash with ice-cold PBS containing phosphatase inhibitors

  • Lyse cells directly in buffer containing:

    • 1% NP-40 or Triton X-100

    • 20 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1 mM EDTA

    • Phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 1 mM sodium pyrophosphate)

    • Protease inhibitor cocktail

• Sample Processing:

  • Scrape cells thoroughly and transfer to microcentrifuge tubes

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

  • Add SDS-PAGE sample buffer and heat at 95°C for 5 minutes

Maintaining phosphorylation status throughout sample preparation is essential, so all steps should be performed quickly and samples kept cold with appropriate phosphatase inhibitors.

What are the recommended protocols for using Phospho-FLT1 (Tyr1213) antibodies in Western blot analysis?

For optimal Western blot analysis using Phospho-FLT1 (Tyr1213) antibodies, follow these detailed recommendations based on published protocols:

Sample Preparation:

• Load 20-50 μg of total protein per lane

• Use freshly prepared samples whenever possible

Gel Electrophoresis:

• Use 7.5% or 4-12% gradient SDS-PAGE gels due to the large size of VEGFR1 (130-140 kDa)

• Run under reducing conditions using standard Laemmli buffer with 5% β-mercaptoethanol or DTT

Transfer:

• Transfer to PVDF membrane (preferred over nitrocellulose for phosphorylated proteins)

• Use wet transfer at 30V overnight at 4°C or 100V for 2 hours with cooling

• Verify transfer efficiency with reversible protein staining

Blocking and Antibody Incubation:

• Block membrane with 5% BSA (not milk, which contains phosphatases) in TBST for 1 hour at room temperature

• Incubate with primary antibody diluted in 5% BSA/TBST:

  • 1:500 - 1:2000 dilution for most antibodies

  • 0.1 μg/mL for affinity-purified antibodies

• Incubate overnight at 4°C with gentle rocking

• Wash 4-5 times with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000 - 1:10000 in 5% BSA/TBST for 1 hour at room temperature

• Wash 4-5 times with TBST, 5 minutes each

Detection:

• Develop using enhanced chemiluminescence (ECL) reagents

• Expected band size: approximately 130-140 kDa

This protocol is optimized for preserving phosphorylation status and achieving specific detection of phospho-VEGFR1 at Tyr1213.

How should researchers troubleshoot weak or absent signals when using Phospho-FLT1 (Tyr1213) antibodies?

When troubleshooting phospho-specific antibody experiments, consider these common issues and solutions:

Problem: No Signal or Weak Signal

Problem: Non-specific Bands or High Background

Problem: Variable Results Between Experiments

By systematically addressing these issues, researchers can improve detection of Phospho-FLT1 (Tyr1213) in their experimental systems.

How can researchers validate the specificity of Phospho-FLT1 (Tyr1213) antibodies?

Validating antibody specificity is crucial for phospho-specific antibodies. For Phospho-FLT1 (Tyr1213) antibodies, implement these validation steps:

• Stimulation/Inhibition Experiments:

  • Compare unstimulated vs. stimulated (VEGF-A or PlGF) conditions

  • Compare phosphorylation with and without specific inhibitors (e.g., D16F7 mAb)

  • Use phosphatase treatment to eliminate phospho-specific signals

• Peptide Competition Assays:

  • Pre-incubate antibody with phospho-peptide immunogen

  • Compare signal with and without peptide competition

  • Signal should be significantly reduced or eliminated in the presence of the specific phosphopeptide

• Genetic Approaches:

  • Use VEGFR1/FLT1 knockdown or knockout models

  • Create Tyr1213 point mutants (Y1213F) to prevent phosphorylation

  • Compare wild-type vs. mutant receptor phosphorylation

• Cross-Validation with Alternative Methods:

  • Confirm phosphorylation using mass spectrometry

  • Use in vitro kinase assays with purified components

  • Employ alternative phospho-specific antibodies from different manufacturers

• Functional Validation:

  • Correlate Tyr1213 phosphorylation with downstream Erk1/2 phosphorylation

  • Demonstrate biological effects of phosphorylation inhibition

These validation approaches provide multiple lines of evidence for antibody specificity and increase confidence in experimental results.

What are the considerations for cross-species applications of Phospho-FLT1 (Tyr1213) antibodies?

When applying Phospho-FLT1 (Tyr1213) antibodies across different species, researchers should consider:

• Sequence Conservation:

  • The region surrounding Tyr1213 shows high conservation across mammals

  • Available antibodies have demonstrated reactivity with human, mouse, and rat samples

  • Some antibodies have broader cross-reactivity including bovine, goat, and sheep samples

• Validation Requirements:

  • Always validate antibody performance in your specific species of interest

  • Check the amino acid sequence surrounding Tyr1213 in your target species

  • Use positive controls from the species being studied

• Species-Specific Considerations:

• Technical Adaptations:

  • Adjust antibody concentrations for different species

  • Optimize blocking conditions to minimize background

  • Consider longer incubation times for less-validated species

  • Perform additional controls when working with non-standard species

By carefully considering these factors, researchers can effectively apply Phospho-FLT1 (Tyr1213) antibodies across different experimental systems and species.

How should researchers design experiments to study dynamic changes in FLT1 Tyr1213 phosphorylation?

To effectively capture dynamic changes in FLT1 Tyr1213 phosphorylation, consider this experimental approach:

• Time-Course Analysis:

  • Establish baseline (unstimulated) conditions

  • Stimulate with VEGF-A or PlGF (50 ng/ml) for multiple timepoints: 0, 2, 5, 10, 15, 30, 60 minutes

  • Process all samples simultaneously using standardized protocols

• Quantification Approach:

  • Normalize phospho-FLT1 (Tyr1213) signal to total FLT1 levels

  • Use image analysis software to quantify band intensities

  • Present data as fold-change relative to baseline

  • Include statistical analysis across multiple experiments

• Experimental Controls:

  • Include positive control (pervanadate-treated cells)

  • Include inhibitor controls (e.g., D16F7 mAb)

  • Maintain consistent cell density and starvation conditions between experiments

• Advanced Analysis Methods:

  • Consider phospho-flow cytometry for single-cell analysis

  • Implement immunofluorescence to observe spatial distribution of phosphorylated receptor

  • Use proximity ligation assays to detect interaction with downstream effectors

This systematic approach allows researchers to capture the temporal dynamics of Tyr1213 phosphorylation and correlate them with downstream signaling events.

What are the implications of Tyr1213 phosphorylation for angiogenesis research?

Understanding Tyr1213 phosphorylation has significant implications for angiogenesis research:

• Mechanistic Insights:

  • Tyr1213 phosphorylation represents a critical step in VEGFR1 activation pathway

  • It provides a specific molecular marker for receptor activation status

  • The phosphorylation leads to ERK1/2 pathway activation, which is central to angiogenic responses

• Therapeutic Relevance:

  • Blocking Tyr1213 phosphorylation (e.g., with D16F7 mAb) prevents downstream signaling

  • This approach may be more effective than targeting individual ligands, as it blocks responses to both VEGF-A and PlGF

  • Combining VEGFR1 phosphorylation inhibitors with other anti-angiogenic approaches may enhance therapeutic efficacy

• Research Applications:

  • Monitoring Tyr1213 phosphorylation can serve as a readout for testing novel anti-angiogenic compounds

  • It allows differentiation between compounds that block receptor-ligand binding versus those that inhibit kinase activity

  • The phosphorylation status can be used to evaluate resistance mechanisms to anti-angiogenic therapies

• Pathological Contexts:

  • VEGFR1 Tyr1213 phosphorylation has been studied in glioblastoma models

  • Similar mechanisms may apply to other cancer types and pathological angiogenesis situations

  • The phosphorylation may play roles beyond angiogenesis, including in inflammatory responses and cell migration

By focusing on this specific phosphorylation event, researchers can develop more targeted approaches to modulating angiogenesis in both experimental and therapeutic contexts.

How does the detection of phosphorylated Tyr1213 differ between various experimental models?

The detection of phosphorylated Tyr1213 varies across experimental models due to differences in VEGFR1 expression levels, activation mechanisms, and technical considerations:

What emerging technologies might improve the detection and analysis of FLT1 Tyr1213 phosphorylation?

Several emerging technologies hold promise for advancing research on VEGFR1/FLT1 Tyr1213 phosphorylation:

• Mass Spectrometry-Based Approaches:

  • Targeted mass spectrometry for absolute quantification of phosphorylated vs. non-phosphorylated peptides

  • Phosphoproteomics for studying global changes in phosphorylation networks following VEGFR1 activation

  • SILAC or TMT labeling for comparative analysis across experimental conditions

• Advanced Microscopy Techniques:

  • Super-resolution microscopy to visualize receptor clustering and colocalization with signaling partners

  • Live-cell FRET-based biosensors to monitor phosphorylation dynamics in real-time

  • Correlative light and electron microscopy to relate phosphorylation events to subcellular structures

• Single-Cell Technologies:

  • Single-cell phospho-proteomics to capture heterogeneity in VEGFR1 signaling

  • Phospho-flow cytometry with improved sensitivity for phospho-epitopes

  • Spatial transcriptomics combined with phospho-protein detection

• Computational Approaches:

  • Machine learning algorithms to predict phosphorylation dynamics from multi-omics data

  • Systems biology modeling of VEGFR1 signaling networks

  • In silico screening for compounds that specifically target Tyr1213 phosphorylation

These technologies promise to provide deeper insights into the spatiotemporal dynamics, regulation, and functional consequences of VEGFR1 Tyr1213 phosphorylation in normal and pathological contexts.

How might understanding of Tyr1213 phosphorylation impact development of targeted therapies?

Understanding the mechanisms and consequences of VEGFR1 Tyr1213 phosphorylation has significant implications for therapeutic development:

• Targeted Inhibition Strategies:

  • Development of compounds specifically blocking Tyr1213 phosphorylation without affecting other receptor functions

  • Design of peptide-based inhibitors mimicking the Tyr1213 region to compete for kinase activity

  • Allosteric modulators that prevent conformational changes required for Tyr1213 phosphorylation

• Dual-Targeting Approaches:

  • Combining Tyr1213 phosphorylation inhibitors with other anti-angiogenic strategies

  • Targeting both VEGFR1 and downstream effectors (e.g., ERK pathway components)

  • Developing bispecific antibodies that simultaneously block ligand binding and phosphorylation

• Biomarker Development:

  • Using Tyr1213 phosphorylation status as a biomarker for therapy selection

  • Monitoring phosphorylation changes to predict resistance to anti-angiogenic therapies

  • Developing companion diagnostics based on phosphorylation patterns

• Translational Considerations:

  • Establishing the relationship between in vitro phosphorylation inhibition and in vivo efficacy

  • Determining optimal biological contexts for targeting this phosphorylation event

  • Developing strategies to overcome potential resistance mechanisms