tyro3 Antibody

What is Tyro3 and what are its primary functions in cellular processes?

Tyro3 (also known as DTK, BYK, RSE, SKY, and TIF) is a receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding to several ligands, including TULP1 and GAS6. It regulates numerous physiological processes including cell survival, migration, and differentiation .

Tyro3 functions through a specific mechanism: ligand binding at the cell surface induces dimerization and autophosphorylation of Tyro3 on its intracellular domain, providing docking sites for downstream signaling molecules . Following activation, Tyro3 interacts with PIK3R1, enhancing PI3-kinase activity and activating the AKT survival pathway, including nuclear translocation of NF-kappa-B and upregulation of NF-kappa-B-regulated genes .

Notably, Tyro3 signaling plays roles in:

• Neuron protection from excitotoxic injury

• Platelet aggregation and cytoskeleton reorganization

• Inhibition of Toll-like receptors (TLRs)-mediated innate immune response by activating STAT1, which selectively induces production of suppressors of cytokine signaling SOCS1 and SOCS3

• Recognition and clearance of apoptotic cells through efferocytosis by macrophages

What methods are recommended for detecting Tyro3 in different experimental systems?

Detection of Tyro3 can be accomplished through several validated methods, depending on your experimental goals:

Western Blot (WB):

• Recommended dilution: 1:500-1:2000

• Positive detection has been confirmed in mouse brain tissue and rat brain tissue

• Observed molecular weight is typically 140 kDa, though the calculated molecular weight is 97 kDa

Immunohistochemistry (IHC):

• Recommended dilution: 1:50-1:500

• Positive staining demonstrated in human colon cancer tissue and mouse brain tissue

• Antigen retrieval suggested with TE buffer pH 9.0, or alternatively with citrate buffer pH 6.0

• For mouse embryo studies, 5 μg/mL overnight at 4°C has shown specific labeling in follicles of vibrissae

Other applications:

• Immunofluorescence (IF) and Flow Cytometry (FC) have been reported in published applications

• ELISA can be used for detecting anti-Tyro3 autoantibodies in serum samples

It's important to note that optimal dilutions should be determined by each laboratory for each specific application to achieve reproducible results.

What are the optimal storage and handling practices for Tyro3 antibodies?

To maintain antibody integrity and activity, follow these storage recommendations:

Long-term storage:

• Store at -20°C to -70°C

• Antibodies are typically stable for 12 months from date of receipt under these conditions

• Avoid repeated freeze-thaw cycles by using a manual defrost freezer

After reconstitution:

• For short-term use (within 1 month): Store at 2-8°C under sterile conditions

• For extended use (up to 6 months): Store at -20°C to -70°C under sterile conditions

• Some formulations contain 0.02% sodium azide and 50% glycerol pH 7.3

Special considerations:

• For smaller aliquots (20μL), some manufacturers include 0.1% BSA in the formulation

• Aliquoting is generally unnecessary for -20°C storage according to some manufacturers

How can researchers effectively validate Tyro3 antibody specificity for experimental use?

Validating antibody specificity is crucial for obtaining reliable experimental results. For Tyro3 antibodies, consider these validation approaches:

Positive controls:

• Use known Tyro3-expressing tissues such as mouse/rat brain tissue for Western blotting

• For IHC, human colon cancer tissue and mouse brain tissue serve as reliable positive controls

Knockout/knockdown validation:

• Generate Tyro3 knockout cells using CRISPR/Cas9 technology to confirm antibody specificity

• Researchers have validated antibody specificity using Tyro3 knockout cells in cancer studies, comparing signals between wild-type and knockout samples

Recombinant protein controls:

• Use recombinant human Tyro3 protein as a positive control in ELISA assays

• Commercial recombinant Tyro3 protein from the wheat germ expression system (fused with GST-tag at N-terminal and purified by glutathione sepharose 4 fast flow) has been successfully used

Multiple antibody validation:

• Compare results using antibodies targeting different epitopes of Tyro3

• Cross-validate findings using different detection methods (WB, IHC, IF, ELISA)

Expected molecular weight verification:

• Confirm detection at the expected molecular weight (observed at approximately 140 kDa in many systems, though calculated MW is 97 kDa)

What are the methodological considerations for purifying anti-Tyro3 antibodies from patient samples?

Purification of anti-Tyro3 antibodies from patient samples, particularly those with autoimmune conditions like SLE, requires careful methodological consideration:

Total IgG isolation:

• Isolate total IgG from patient serum using thiophilic adsorbent reagent according to manufacturer's instructions

• Concentrate the isolated IgG in a centrifuge tube (e.g., Amicon, Millipore)

Specific anti-Tyro3 antibody purification:

• Use AminoLink Plus Coupling Resin for purification of specific antibodies

• Couple recombinant human Tyro3 protein to the resin

• Incubate the purified total IgG from patients with the resin-bound complex using gentle end-over-end mixing

• Elute the antibody and neutralize with 1M Tris (pH=9.0)

• Perform sterile filtration and store as small aliquots at -80°C

This method has been successfully employed to study the effects of purified anti-Tyro3 antibodies on macrophage efferocytosis in SLE research .

How does Tyro3 signaling affect macrophage efferocytosis in autoimmune diseases?

Tyro3 plays a critical role in macrophage efferocytosis (clearance of apoptotic cells), with significant implications for autoimmune diseases:

Mechanism of action:

• Tyro3 functions as a receptor responsible for the recognition of apoptotic cells during efferocytosis by macrophages

• It belongs to the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases that are crucial for macrophage efferocytosis

Impact in autoimmune diseases:

• In SLE, research has demonstrated that autoantibodies against Tyro3 inhibit macrophage efferocytosis

• Studies using flow cytometry and immunofluorescence have shown that purified anti-Tyro3 IgG from SLE patients significantly inhibits the efferocytosis capability of macrophages (p = 0.004 and 0.044, respectively) compared with unconjugated human IgG

Clinical correlation:

• Serum levels of anti-Tyro3 IgG are significantly elevated in patients with SLE compared to rheumatoid arthritis, primary Sjögren's Syndrome, and healthy controls (all p < 0.0001)

• Anti-Tyro3 IgG levels positively correlate with SLE disease activity index (SLEDAI) score (r = 0.254, p = 0.034), suggesting a relationship between these antibodies and disease severity

This mechanism provides insight into how defects in efferocytosis contribute to the pathogenesis of SLE through accumulation of apoptotic debris and subsequent immune dysregulation.

What is the role of Tyro3 in cancer immunotherapy resistance?

Tyro3 has emerged as a significant factor in resistance to immune checkpoint blockade therapies:

Mechanistic insights:

• Tumors with high TYRO3 expression exhibit resistance to anti-PD-1/PD-L1 therapy in both syngeneic mouse models and patients receiving immunotherapy

• TYRO3 inhibits tumor cell ferroptosis triggered by anti-PD-1/PD-L1 therapy

• TYRO3 promotes a protumor microenvironment by reducing the M1/M2 macrophage ratio, contributing to therapy resistance

Experimental evidence:

• In mouse models, Tyro3 overexpression in anti-PD-1-responsive tumors (4T1-P) renders them resistant to treatment

• Conversely, knockout of Tyro3 in resistant tumors can restore sensitivity to anti-PD-1 therapy

Signaling pathway:

• TYRO3-dependent CCN1 secretion has been identified as mediating immune-suppressive reactions via tumor-associated macrophages (TAMs)

• Inhibition of the TYRO3-CCN1 axis can boost antitumor immune responses in both tumor-draining lymph nodes and tumors in mouse models

Therapeutic implications:

• Pharmacological blockade of TYRO3 using selective inhibitors (such as KRCT87) can enhance responsiveness to anti-PD-1 therapy, even in models that typically don't respond

• The combination of TYRO3 inhibitors and anti-PD-1 therapy shows synergistic antitumor effects in otherwise resistant tumors

How do Tyro3 knockout or inhibition models affect immune responses in vivo?

TAM receptor knockout phenotypes:

• Triple knockout mice lacking all three TAM receptors (Tyro3, Axl, Mer) exhibit mild thrombocytopenia with platelet counts less than 50% of control mice

• Single or double knockout mice for TAM receptors maintain normal platelet counts in peripheral blood

Bleeding time and platelet function:

• Mice lacking any combination of two receptors (double mutants) display prolonged bleeding times despite normal platelet counts, suggesting platelet dysfunction rather than impaired biogenesis

• Triple mutant mice (lacking all three receptors) show the most severe platelet function abnormality, contributing to increased bleeding time

Megakaryocytopoiesis phenotypes:

• Different combinations of double mutant mice (TA, AM, or TM) display distinct megakaryocytopoiesis phenotypes despite normal platelet levels

• There's a notable inconsistency between the marked defect in proplatelet formation and the relatively mild decrease in peripheral blood platelet count in TAM mice

Immune modulation effects:

• TYRO3 blockade enhances antitumor immune responses in mouse models

• Inhibition of TYRO3 fosters macrophage polarization toward M1-skewing phenotypes, triggering antitumor T-cell responses

• These effects are mediated through inhibition of TYRO3-driven CCN1 secretion

What factors can affect the results of Tyro3 detection in experimental systems?

Several technical factors can influence the reliability of Tyro3 detection:

Antibody selection considerations:

• Different antibodies may recognize different epitopes, affecting detection sensitivity

• Polyclonal vs. monoclonal antibodies: Polyclonal antibodies like 28513-1-AP recognize multiple epitopes and may provide stronger signals but potentially less specificity compared to monoclonals like EPR4308

Sample preparation factors:

• For IHC, antigen retrieval method is critical: TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used alternatively

• For WB, the observed molecular weight (140 kDa) differs significantly from calculated weight (97 kDa), suggesting post-translational modifications that should be considered when interpreting results

Detection system variables:

• For ELISA detection of anti-Tyro3 antibodies, blocking with PBS-T containing 5% BSA for 2 hours at 37°C is recommended

• For optimal secondary antibody selection, HRP-conjugated goat anti-human IgG has been successfully used with incubation for 1 hour at room temperature

Experimental controls:

• Include recombinant human Tyro3/Dtk antibody as a positive control in ELISA assays

• Use known positive tissue samples (mouse/rat brain tissue, human colon cancer tissue) as controls

How can researchers resolve discrepancies between Tyro3 antibody detection methods?

When facing inconsistent results across different detection methods:

Cross-validation approach:

• Confirm findings using at least two different detection methods (e.g., WB and IHC)

• If inconsistencies persist, use genetic approaches (siRNA knockdown or CRISPR knockout) to validate specificity

• Consider using different antibodies targeting distinct epitopes of Tyro3

Method-specific considerations:

• For Western blotting: Post-translational modifications may affect detection, explaining the discrepancy between calculated (97 kDa) and observed (140 kDa) molecular weights

• For IHC/IF: Background staining can be reduced by optimizing blocking conditions and antibody dilutions

• For ELISA: Consider cross-reactivity with other TAM family members (Axl, Mer) when interpreting results

Quantification standardization:

• Use recombinant Tyro3 protein to generate standard curves for quantitative assessments

• When comparing results across methods, focus on relative changes rather than absolute values

• Include appropriate housekeeping genes or loading controls for normalization

How are anti-Tyro3 antibodies used to study autoimmune disease mechanisms?

Anti-Tyro3 antibodies serve as valuable tools for investigating autoimmune pathogenesis:

Autoantibody detection in clinical samples:

• ELISA assays using recombinant human Tyro3 protein can detect autoantibodies against Tyro3 in patient sera

• This approach revealed that anti-Tyro3 IgG levels are significantly elevated in SLE patients compared to other autoimmune conditions and healthy controls

Clinical correlations:

• Anti-Tyro3 IgG levels positively correlate with:

  • SLE disease activity index (SLEDAI) score (r = 0.254, p = 0.034)

  • Erythrocyte sedimentation rate (ESR) (r = 0.430, p < 0.001)

  • C-reactive protein (CRP) (r = 0.246, p = 0.049)

  • Immunoglobulin G (IgG) (r = 0.408, p = 0.001)

• They negatively correlate with hemoglobin (Hb) (r = −0.294, p = 0.014)

Functional studies:

• Purified anti-Tyro3 antibodies from SLE patients can be used to determine their effects on macrophage efferocytosis

• Flow cytometry and immunofluorescence have demonstrated that these antibodies inhibit macrophage efferocytosis, potentially contributing to disease pathogenesis

Diagnostic potential:

• ROC curve analysis indicates that anti-Tyro3 antibodies can differentiate SLE patients from healthy controls

• Anti-Tyro3 antibodies were found in 30.2% of anti-Sm-negative patients and 28.6% of anti-dsDNA-negative patients, suggesting potential utility as a complementary diagnostic marker

What methodological approaches are used to study Tyro3's role in cancer immunotherapy resistance?

To investigate Tyro3's contribution to immunotherapy resistance, researchers employ several methodological approaches:

Receptor tyrosine kinase (RTK) profiling:

• Commercial RTK antibody arrays can identify differential expression or phosphorylation of Tyro3 between immunotherapy-responsive and resistant cancer cells

• This approach identified TYRO3 as having the highest increase in expression in anti-PD-1-resistant 4T1 cells compared to responsive cells

Genetic manipulation strategies:

• Overexpression models: Lentiviral infection can be used to overexpress Tyro3 in responsive cancer cells to determine if this induces resistance

• Knockout models: CRISPR/Cas9-mediated Tyro3 knockout in resistant cells can determine if this restores therapy sensitivity

In vivo therapeutic evaluations:

• Syngeneic mouse models implanted with Tyro3-manipulated tumor cells can assess responses to anti-PD-1 therapy

• Parameters measured include tumor growth kinetics and survival curves to quantify treatment effects

Macrophage polarization assessment:

• Co-culture systems with THP1 cells can evaluate M1/M2 macrophage polarization

• Measure mRNA expression of M1 markers (IL6, CXCL10, HLADRA1) and M2 markers (CD206/MRC1, ARG1, IL10) to assess polarization status

• This approach revealed that conditioned media from Tyro3-overexpressing cells decreased M1 markers and increased M2 markers

Signaling pathway analysis:

• Investigate the TYRO3-CCN1 axis by measuring CCN1 secretion and its effects on the tumor microenvironment

• Assess ferroptosis markers in tumor cells following TYRO3 inhibition to understand resistance mechanisms

What are the key considerations for designing studies to evaluate Tyro3 inhibitors?

When designing studies to evaluate Tyro3 inhibitors, researchers should consider:

Inhibitor selection and characterization:

• Assess potency and selectivity against other TAM family members (Axl and Mer)

• For example, KRCT87 was identified through screening a 208-compound TYRO3-focused chemical library and characterized as a potent and highly selective TYRO3 inhibitor

Model system selection:

• Choose appropriate syngeneic tumor models that express Tyro3 at different levels

• MC38 and 4T1 tumor models have been successfully used to evaluate Tyro3 inhibitors

Combination therapy approaches:

• Design studies to evaluate both monotherapy and combination approaches

• The combination of TYRO3 inhibitors with anti-PD-1 therapy has shown synergistic effects, particularly in models normally non-responsive to immunotherapy alone

Mechanistic readouts:

• Include assays that measure macrophage polarization (M1/M2 ratio)

• Assess T-cell responses and activation markers

• Evaluate CCN1 secretion and its downstream effects

Translational considerations:

• Include biomarker analyses to identify potential predictors of response

• Consider evaluating TYRO3 expression levels in patient samples as a stratification approach

What are the emerging applications of Tyro3 antibodies in cancer research?

Recent advances have expanded the potential applications of Tyro3 antibodies in cancer research:

Biomarker development:

• Tyro3 expression is being investigated as a potential biomarker for immunotherapy resistance

• High TYRO3 expression has been associated with anti-PD-1/PD-L1 resistance in both mouse models and patient samples

Therapeutic targeting:

• Development of neutralizing antibodies against Tyro3 could provide therapeutic benefits

• Anti-Tyro3 approaches might synergize with existing immunotherapies to overcome resistance

Tumor microenvironment modulation:

• Tyro3 antibodies are valuable tools for studying how Tyro3 signaling shapes the tumor immune landscape

• Research shows Tyro3 influences macrophage polarization and T-cell activity in the tumor microenvironment

Combination therapy design:

• Understanding Tyro3's role in treatment resistance is guiding rational combination strategies

• The TYRO3-CCN1 axis has emerged as a promising target to enhance immunotherapy responses

Ferroptosis regulation:

• Recent discoveries link Tyro3 to ferroptosis resistance in tumors

• Antibodies targeting Tyro3 can help elucidate this mechanism and potentially guide ferroptosis-inducing therapeutic strategies

How does Tyro3 function in the context of viral infections?

Recent research has revealed important roles for Tyro3 in viral infections:

Viral entry receptor:

• Tyro3 can function as a receptor for several viruses, including:

  • Lassa virus

  • Lymphocytic choriomeningitis virus

  • Ebolavirus

Mechanism of viral recognition:

• These interactions likely occur through GAS6 (a Tyro3 ligand) binding to phosphatidylserine on the surface of the virion envelope

• This mechanism resembles how Tyro3 recognizes apoptotic cells during efferocytosis

Therapeutic implications:

• Tyro3 antibodies could potentially serve as tools to study viral entry mechanisms

• Blocking Tyro3 might represent a therapeutic strategy against certain viral infections

Immune modulation during infection:

• Given Tyro3's role in inhibiting Toll-like receptor (TLR)-mediated innate immune responses, its signaling might influence antiviral immunity

• Tyro3 activates STAT1, which selectively induces production of suppressors of cytokine signaling SOCS1 and SOCS3