KDR/FLT4 Antibody

What are KDR and FLT4 and what is their significance in vascular biology?

KDR (Kinase insert Domain Receptor, also known as VEGFR2, FLK1, or CD309) is a receptor tyrosine kinase that acts as a cell-surface receptor for VEGFA, VEGFC, and VEGFD. In humans, KDR has a canonical length of 1356 amino acid residues and a mass of approximately 151.5 kDa . Its subcellular localization includes cytoplasmic vesicles, endoplasmic reticulum, and cell membrane.

FLT4 (Fms-related Tyrosine kinase 4, also known as VEGFR3) is another receptor tyrosine kinase that plays a critical role in lymphatic vessel development and maintenance . It is predominantly expressed in lymphatic endothelial cells but can also be found in some blood vessels during development.

Their significance stems from their fundamental roles in:

• KDR: Primarily mediates angiogenesis (blood vessel formation)

• FLT4: Primarily regulates lymphangiogenesis (lymphatic vessel formation)

Both receptors are critical in developmental biology, cancer research, and vascular pathology studies.

How do KDR and FLT4 expression patterns differ across tissue types?

Expression patterns of KDR and FLT4 vary considerably across tissues:

KDR is:

• Notably expressed in cornea

• Highly expressed in vascular endothelial cells

• Often upregulated in tumor vasculature

• Present in human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs)

FLT4 is:

• Predominantly expressed in lymphatic endothelial cells

• Detected in human umbilical vein endothelial cells (HUVECs)

• Present in mouse liver (used as a positive control in antibody validation)

• Expressed in certain leukemia cell lines but rarely in lymphoma cell lines

Research shows differential expression in leukemic entities: approximately 16% (10/62) of leukemia cell lines expressed KDR, while 42% (27/65) expressed FLT4. In contrast, lymphoma cell lines rarely express either receptor - none of thirty lymphoma cell lines showed significant KDR expression, and only one showed FLT4 expression .

What epigenetic mechanisms regulate KDR and FLT4 expression?

DNA methylation plays a crucial role in regulating KDR and FLT4 expression, as demonstrated by several lines of evidence:

• Methylation status correlates with expression:

  • CpG islands in the promoter regions of KDR and FLT4 are unmethylated in HUVECs, HDMECs, and in KDR+ and FLT4+ cell lines

  • Methylated promoters in KDR- and FLT4- cell lines correlate with silenced expression

• Demethylating treatment effects:

  • The demethylating agent 5-Aza-2'deoxycytidine (5-Aza-dC) induces expression of KDR and FLT4 in methylated cell lines but not in unmethylated cell lines

  • This provides direct evidence of epigenetic regulation

• Quantifiable correlation:

  • The accuracy of methylation-specific PCR (M-PCR) for predicting KDR mRNA expression (threshold >0.1) is approximately 88%

  • The accuracy of M-PCR for predicting FLT4 mRNA expression (threshold >0.1) is approximately 80%

These findings suggest that epigenetic modifications represent an important regulatory mechanism controlling the expression of these vascular receptors in different cell types.

What criteria should researchers use when selecting KDR or FLT4 antibodies?

When selecting antibodies against KDR or FLT4, researchers should consider the following criteria:

• Target specificity:

  • Verify whether the antibody recognizes total protein or phosphorylated forms

  • For phospho-specific studies, select antibodies targeting relevant phosphorylation sites (e.g., Y1054/Y1063 for FLT4)

  • Check if the antibody recognizes specific isoforms (up to 3 different isoforms have been reported for KDR)

• Species reactivity:

  • Confirm species cross-reactivity (many antibodies react with human, mouse, and rat)

  • Note that species-specific optimization may be required even for cross-reactive antibodies

• Validated applications:

  • Select antibodies validated for your specific application (WB, IHC, IF, ELISA, FACS)

  • Common applications for KDR/FLT4 antibodies include Western Blot, ELISA, Flow Cytometry, and Immunohistochemistry

• Immunogen information:

  • Check the immunogen sequence to understand the epitope region

  • Example: Some FLT4 antibodies are generated against amino acids 330-553 of human VEGFR3/FLT4 or Y25-N259

• Validation data:

  • Review validation data in relevant tissues (e.g., mouse liver for FLT4)

  • Check for cross-reactivity with related proteins (e.g., other VEGF receptors)

• Recommended dilutions:

  • Note the suggested working dilutions for each application (e.g., for FLT4: WB 1:100-1:500, IHC-P 1:50-1:100, IF/ICC 1:50-1:200)

How can researchers validate the specificity of KDR and FLT4 antibodies?

Validating antibody specificity is critical for reliable research outcomes. For KDR and FLT4 antibodies, consider these validation strategies:

• Positive and negative controls:

  • Positive tissue controls: Use tissues with known expression (e.g., mouse liver for FLT4)

  • Positive cell controls: Use cells with confirmed expression (e.g., HUVECs and HDMECs for both KDR and FLT4)

  • Negative controls: Use tissues or cell lines with confirmed absence of expression

• Correlation with transcript data:

  • Compare antibody detection with qPCR data for the same samples

  • The search results provide a correlation table between mRNA expression and protein detection

• Multiple antibody validation:

  • Use multiple antibodies targeting different epitopes of the same protein

  • Compare staining patterns to confirm consistency

• Knockdown/knockout validation:

  • Use siRNA, shRNA, or CRISPR-based approaches to reduce target expression

  • Observe corresponding reduction in antibody signal

• Phospho-specificity validation:

  • For phospho-specific antibodies, treat samples with phosphatases

  • Validate signal increase after receptor stimulation (e.g., with VEGF-C for FLT4)

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide

  • Observe elimination of specific signal

• Cross-reactivity assessment:

  • Test on closely related family members to confirm specificity

  • For example, human VEGFR3/FLT4 antibody showed approximately 15% cross-reactivity with recombinant mouse VEGFR3 in Western blots

What are the optimal protocols for detecting KDR/FLT4 using immunohistochemistry?

For optimal immunohistochemical detection of KDR/FLT4, follow these protocol guidelines:

Sample Preparation:

• Fixation: Use freshly prepared 4% paraformaldehyde for consistent results

• Sectioning: 4-6 μm thick sections for FFPE tissue; 8-10 μm for frozen sections

• Antigen retrieval: Heat-mediated retrieval in citrate buffer (pH 6.0) for 20 minutes is recommended for FLT4

Staining Protocol:

• Blocking: 5-10% normal serum (from secondary antibody species) with 1% BSA for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution: 1:50-1:100 for IHC-P for FLT4 antibodies

  • Incubation: Overnight at 4°C for optimal sensitivity

• Secondary antibody incubation: 1-2 hours at room temperature

• Detection system: HRP-DAB for chromogenic detection; fluorescent secondaries for IF

• Counterstaining: Hematoxylin for brightfield; DAPI for fluorescence

Controls and Validation:

• Include positive controls (e.g., lymphatic vessels for FLT4)

• Include negative controls (primary antibody omission)

• Consider dual staining with endothelial markers (CD31) to confirm vascular localization

Visualization:

• For complex vascular networks, consider maximum intensity projection of confocal images

• For co-localization studies, sequential scanning reduces bleed-through

What methodologies are recommended for studying KDR/FLT4 protein-protein interactions?

Several methodologies are particularly effective for studying KDR/FLT4 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Useful for identifying physical interactions between KDR/FLT4 and binding partners

  • Example from research: LEC whole cell lysate was immunoprecipitated with anti-PI3K followed by Western blotting for VEGFR-3 to demonstrate direct interaction

  • Treatment conditions: Vehicle control, IgG control, VEGF-C (100 ng/ml), or VEGF-A (100 ng/ml) for 15 minutes prior to lysis

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein interactions in situ with high sensitivity

  • Research example: Detection of VEGFR-3/phospho-PI3K complexes in LECs with and without VEGF-C stimulation

  • Quantification: PLA signals can be quantified using specialized imaging software

• FRET/BRET analysis:

  • For studying dynamic interactions in living cells

  • Requires fusion proteins with appropriate fluorescent/bioluminescent tags

• Cross-linking mass spectrometry:

  • For mapping interaction interfaces at amino acid resolution

  • Particularly useful for characterizing novel binding partners

• Receptor dimerization assays:

  • Specific techniques to study homodimerization (KDR-KDR, FLT4-FLT4) or heterodimerization (KDR-FLT4)

  • Often combined with functional readouts (phosphorylation, downstream signaling)

• Functional validation of interactions:

  • Monitor downstream signaling (p42/44 MAPK activation) following VEGF-C stimulation

  • Cell-based assays to assess biological consequences of disrupting specific interactions

How can phosphorylation-specific antibodies be used to study KDR/FLT4 activation?

Phosphorylation-specific antibodies are valuable tools for studying the activation state of KDR/FLT4 receptors:

• Detection of activation status:

  • Phospho-specific antibodies like anti-phospho-Flk-1/Flt-4 (Y1054/Y1063) detect active receptor forms

  • These antibodies recognize specific phosphorylated tyrosine residues that indicate receptor activation

• Stimulation protocols:

  • Stimulate cells with appropriate ligands: VEGF-A for KDR, VEGF-C for FLT4

  • Optimal stimulation time: 15 minutes for detecting VEGFR-3/phospho-PI3K complexes

  • Concentration: 100 ng/ml VEGF-C has been shown effective for FLT4 activation

• Downstream signaling assessment:

  • Monitor p42/44 MAPK activation following receptor phosphorylation

  • Example: VEGF-C induced p42/44 MAPK activation in the KDR-/FLT4+ cell line OCI-AML1

• Quantification approaches:

  • Western blot: Quantify band intensity normalized to total receptor expression

  • Flow cytometry: Measure phospho-specific antibody binding at single-cell resolution

  • Immunofluorescence: Analyze subcellular localization of phosphorylated receptors

• Inhibitor studies:

  • Combine with receptor tyrosine kinase inhibitors to confirm specificity

  • Use phosphatase inhibitors (sodium orthovanadate, sodium fluoride) during sample preparation to preserve phosphorylation

• Multiplexed analysis:

  • Combine phospho-KDR/FLT4 detection with markers of downstream pathway activation

  • Co-stain for internalization markers to assess receptor trafficking upon activation

How can researchers utilize KDR/FLT4 antibodies to study lymphangiogenesis in development and disease?

KDR/FLT4 antibodies can be strategically employed to investigate lymphangiogenesis across various developmental and pathological contexts:

• Developmental lymphangiogenesis:

  • Whole-mount immunohistochemistry to visualize lymphatic vessel development in embryonic tissues

  • Example: Analysis of lymphatic vessels in the dorsal skin of E16.5 and E18.5 embryos using VEGFR3/FLT4 antibodies

  • Quantification parameters: Distance between migrating fronts and vessel diameter

• Lymphatic vessel maintenance:

  • Identify valve formation in collecting lymphatic vessels

  • Detect LVs (lymphatic valves) using FLT4 staining in collecting vessels

  • Compare normal versus pathological conditions (e.g., lymphedema models)

• Tumor lymphangiogenesis:

  • Dual staining with blood vessel markers to differentiate lymphatic from blood vessels

  • Quantify peritumoral and intratumoral lymphatic vessel density

  • Correlate with metastatic potential

• Inflammation-induced lymphangiogenesis:

  • Track expansion of lymphatic networks during inflammation

  • Compare with pro-inflammatory markers to establish temporal relationships

• Lymphatic endothelial cell isolation and characterization:

  • Flow cytometry-based sorting using FLT4 antibodies

  • Culture and characterize primary lymphatic endothelial cells (LECs)

  • Example: Lymphatic origin of murine LECs was confirmed by IHC for Prox-1, VEGFR3, and Podoplanin

• Genetic manipulation studies:

  • Assess phenotypic consequences of genetic alterations (e.g., Lyve1-Cre;Yapf/f;Tazf/f embryos)

  • Combine with lineage tracing approaches

• Therapeutic targeting assessment:

  • Monitor effects of anti-lymphangiogenic therapies on FLT4 expression and signaling

  • Identify compensatory mechanisms after pathway inhibition

What experimental approaches combine KDR/FLT4 antibodies with genetic manipulation techniques?

Integrating KDR/FLT4 antibodies with genetic manipulation techniques creates powerful experimental paradigms:

• Conditional knockout validation:

  • Use KDR/FLT4 antibodies to confirm tissue-specific deletion in conditional knockout models

  • Example: Analyzing lymphatic vessel abnormalities in Lyve1-Cre;Yapf/f;Tazf/f embryos using VEGFR3 antibodies

  • Quantify phenotypic parameters: vessel diameter, branching, and network formation

• Promoter activity studies:

  • Combine reporter gene constructs (driven by KDR/FLT4 promoters) with antibody staining

  • Correlate endogenous protein expression with promoter activity

  • Investigate the effects of DNA methylation status on expression

• CRISPR/Cas9-mediated editing:

  • Introduce specific mutations in receptor tyrosine phosphorylation sites

  • Use phospho-specific antibodies to validate functional consequences

  • Assess effects on downstream signaling (p42/44 MAPK activation)

• Rescue experiments:

  • Reintroduce wild-type or mutated receptors in knockout backgrounds

  • Use antibodies to confirm expression and localization

  • Evaluate functional recovery

• Chimeric receptor studies:

  • Create KDR/FLT4 chimeric receptors to study domain-specific functions

  • Use domain-specific antibodies to verify expression and activation

• RNA interference approaches:

  • Validate knockdown efficiency at protein level using KDR/FLT4 antibodies

  • Analyze phenotypic consequences of receptor depletion

  • The inverse approach: demethylating agent 5-Aza-dC induced expression of KDR and FLT4 in methylated cell lines

• Single-cell analysis:

  • Combine FACS sorting based on KDR/FLT4 expression with single-cell RNA-seq

  • Identify subpopulations with distinct molecular signatures

How can researchers investigate cross-talk between KDR and FLT4 signaling pathways?

Investigating cross-talk between KDR and FLT4 signaling pathways requires sophisticated experimental approaches:

• Co-expression analysis:

  • Dual immunostaining to identify cells expressing both receptors

  • Flow cytometric analysis to quantify co-expression at single-cell level

  • Example from research: Cell lines HEL expressed both KDR and FLT4

• Sequential stimulation experiments:

  • Stimulate with VEGF-A (KDR ligand) followed by VEGF-C (FLT4 ligand) or vice versa

  • Monitor receptor phosphorylation and downstream pathway activation

  • Compare responses to simultaneous versus sequential stimulation

• Receptor heterodimerization:

  • Co-immunoprecipitation to detect KDR-FLT4 complexes

  • Proximity ligation assays to visualize heterodimers in situ

  • Analysis of ligand-induced changes in heterodimerization

• Selective inhibition:

  • Use receptor-specific blocking antibodies or small molecule inhibitors

  • Assess effect of inhibiting one receptor on the signaling capacity of the other

  • Monitor compensatory upregulation mechanisms

• Downstream signaling convergence:

  • Investigate shared signaling components (e.g., PI3K pathway)

  • Example: Detection of VEGFR-3/phospho-PI3K complexes after VEGF-C stimulation

  • Compare phosphorylation patterns of common downstream targets

• Genetic manipulation:

  • Create cells with defined KDR/FLT4 expression patterns through CRISPR-based approaches

  • Use phospho-specific antibodies to monitor pathway activation in different genetic backgrounds

• Computational modeling:

  • Integrate experimental data into mathematical models of receptor cross-talk

  • Test predictions using antibody-based validation experiments

What are common challenges in detecting KDR/FLT4 in tissue samples and how can they be overcome?

Detecting KDR/FLT4 in tissue samples presents several challenges with specific solutions:

How can researchers optimize Western blot protocols for detecting KDR and FLT4?

Optimizing Western blot protocols for KDR and FLT4 detection requires attention to several technical considerations:

• Sample preparation:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) to preserve phosphorylation status

  • Add protease inhibitors to prevent degradation

  • Consider non-reducing conditions for certain epitopes

  • Example: For phospho-Flk-1/Flt-4 (Y1054/Y1063) detection

• Gel selection and transfer:

  • Use lower percentage gels (6-8%) for these high molecular weight proteins (KDR: ~151.5 kDa )

  • Extend transfer time for large proteins (overnight at low voltage or 2-3 hours at higher voltage)

  • Consider wet transfer systems for more efficient transfer of large proteins

• Blocking optimization:

  • Test different blocking agents (5% BSA often works better than milk for phospho-specific antibodies)

  • Blocking duration: 1-2 hours at room temperature or overnight at 4°C

• Antibody incubation:

  • Dilution ranges: 1:100-1:500 for FLT4 antibodies in Western blot applications

  • Incubation time: Overnight at 4°C for primary antibody

  • Include 0.1% Tween-20 in antibody diluent to reduce background

• Detection system:

  • For phospho-specific detection, chemiluminescent substrates with higher sensitivity are recommended

  • Consider fluorescent secondary antibodies for multiplexing and quantitative analysis

• Controls and normalization:

  • Include positive control lysates (e.g., HUVECs, HDMECs for both KDR and FLT4)

  • Use appropriate loading controls (vinculin has been used successfully)

  • For phospho-specific studies, always compare to total protein levels

• Troubleshooting specific issues:

  • Multiple bands: May represent different glycosylation states or isoforms (up to 3 isoforms have been reported for KDR)

  • Weak signal: Consider enrichment via immunoprecipitation before Western blot

  • High background: More stringent washing or further antibody dilution

What considerations are important when using KDR/FLT4 antibodies for flow cytometry?

When using KDR/FLT4 antibodies for flow cytometry, consider these important factors:

• Sample preparation:

  • Gentle dissociation techniques to preserve surface epitopes

  • For tissue samples, use collagenase digestion optimized for endothelial cells

  • Maintain cells at 4°C throughout to prevent receptor internalization

• Staining protocol:

  • Surface staining: Incubate cells with antibodies for 30 minutes at 4°C

  • Include viability dye (e.g., propidium iodide ) to exclude dead cells

  • Use Fc receptor blocking to reduce non-specific binding

• Antibody selection:

  • Choose flow cytometry-validated antibodies (e.g., anti-Human CD309/KDR/VEGFR-2 antibody (1121B) for flow cytometry )

  • Consider directly conjugated antibodies to eliminate secondary antibody steps

  • When using unconjugated primary antibodies, select appropriate fluorochrome-conjugated secondaries

• Controls:

  • Include isotype-matched control antibodies

  • Use FMO (fluorescence minus one) controls for multicolor panels

  • Include known positive (HUVECs, HDMECs) and negative cell types

• Instrument settings:

  • Perform proper compensation when using multiple fluorochromes

  • Optimize voltage settings based on positive and negative controls

  • Consider cell size and complexity when analyzing endothelial populations

• Analysis considerations:

  • Gate on viable single cells before analyzing receptor expression

  • For rare populations (e.g., circulating endothelial cells), collect sufficient events

  • Consider co-staining with additional endothelial markers (e.g., CD31 )

• Special applications:

  • For intracellular phospho-epitopes, use appropriate fixation and permeabilization reagents

  • For quantitative analysis, consider using calibration beads

  • For cell sorting, optimize for both purity and viability