PTK2B Antibody

What is PTK2B and why is it significant in research?

PTK2B (Protein Tyrosine Kinase 2 Beta), also known as PYK2, FAK2, RAFTK, and CADTK, is a cytoplasmic tyrosine kinase involved in calcium-induced regulation of ion channels and activation of the MAP kinase signaling pathway. This 116 kDa protein (1009 amino acids) plays crucial roles in:

• Reorganization of the actin cytoskeleton

• Cell polarization, migration, adhesion, and spreading

• Bone remodeling

• Humoral immune response regulation

• Signaling downstream of multiple receptor types (integrin, collagen, immune, G-protein coupled, cytokine, chemokine, and growth factor receptors)

• Cellular stress responses

PTK2B has gained significant research attention due to its implications in Alzheimer's disease as a risk factor, cancer progression (including gliomas, hepatocellular carcinoma, lung cancer, and breast cancer), antiviral immunity, and inflammatory conditions like ulcerative colitis .

What applications are PTK2B antibodies validated for?

PTK2B antibodies are validated for multiple research applications, with specific validation parameters depending on the antibody clone and manufacturer:

Most antibodies show reactivity with human, mouse, rat samples, with some also validated for pig samples .

How do I select the appropriate PTK2B antibody for my specific research question?

Selecting the appropriate PTK2B antibody requires consideration of multiple parameters to ensure experimental success:

• Target specificity: Determine which specific region or phosphorylation site of PTK2B you need to detect:

  • Total PTK2B detection (e.g., 67141-1-Ig targets whole protein)

  • Phosphorylation-specific antibodies (e.g., pTyr402, pTyr579)

• Species reactivity: Verify the antibody's validated reactivity matches your experimental model:

  • Human: Most antibodies work well with human samples

  • Mouse/Rat: Critical for animal models of disease

  • Others: Limited options for other species

• Application compatibility: Ensure the antibody is validated for your required technique:

  • For mechanical studies: WB-validated antibodies

  • For localization: IHC/IF-validated antibodies

  • For protein interactions: IP-validated antibodies

• Antibody type:

  • Monoclonal: Greater specificity, consistent lot-to-lot performance (e.g., 67141-1-Ig)

  • Polyclonal: Higher sensitivity, recognizes multiple epitopes (e.g., 17592-1-AP)

• Validation data: Review the manufacturer's validation data, including:

  • Positive control tissues/cells (e.g., Jurkat cells, brain tissue)

  • Expected molecular weight bands

  • Images demonstrating specificity

For multiple detection methods, consider a well-validated antibody across applications or use complementary antibodies optimized for each technique.

How can I effectively design experiments to study PTK2B phosphorylation dynamics in response to cellular stressors?

Designing experiments to study PTK2B phosphorylation dynamics requires a systematic approach:

• Establish baseline and stimulus conditions:

  • Viral infection models: HSV-1-GFP or VSVΔM51-GFP have been validated for PTK2B phosphorylation studies

  • Inflammatory stimuli: TNF-α treatment activates PTK2B in neutrophils

  • DNA/RNA stimulants: HT-DNA or poly(I:C) for mimicking pathogen exposure

• Design time-course experiments:

  • Capture early phosphorylation events (5-30 min) and sustained changes (1-24 hours)

  • Include appropriate controls (unstimulated, vehicle-treated)

  • Consider parallel assessment of upstream regulators and downstream targets

• Detection strategy:

  • Western blotting: Use phospho-specific antibodies (pTyr402, pTyr579) alongside total PTK2B

  • Include key downstream signaling molecules:

    • For antiviral responses: phospho-TBK1 (at Tyr591), phospho-IRF3, phospho-STING

    • For inflammatory responses: MAPK pathway components

• Pharmacological manipulation:

  • PTK2B inhibitors (e.g., TAE226) to confirm specific roles

  • Pathway inhibitors to delineate signaling hierarchy

  • Phosphatase inhibitors during sample preparation to preserve phosphorylation status

• Genetic approaches for validation:

  • PTK2B knockdown/knockout: shRNA, CRISPR-Cas9 (as described in result )

  • Rescue experiments with wild-type vs. phospho-site mutants

  • Consider tissue-specific knockout models for in vivo studies

For antiviral responses specifically, follow the validated protocol in result , which demonstrated that PTK2B directly phosphorylates TBK1 at Tyr591, promoting oligomerization and activation of both TBK1 and STING through distinct mechanisms.

What are the optimal protocols for studying PTK2B's role in neuronal function and Alzheimer's disease pathology?

Based on recent research identifying PTK2B as an Alzheimer's disease risk factor, the following optimized protocols can be employed:

• Brain tissue processing and analysis:

  • Immunohistochemistry: Use formalin-fixed, paraffin-embedded sections with antigen retrieval (TE buffer pH 9.0)

  • Optimal antibodies: Anti-Pyk2 (Cell Signaling #3480S or Santa Cruz #SC130077) and anti-pPyk2 Y402 (Abcam #ab131543)

  • Dilution range: 1:50-1:500 for IHC applications

  • Pattern analysis: Focus on neuronal and glial cells in cerebral cortex and hippocampus where moderate positivity is expected

• Synaptic function assessment:

  • Electrophysiology: Record field potentials in hippocampal Schaffer collateral pathway

  • Key parameters: Monitor both LTP and LTD, as PTK2B specifically affects LTD induction

  • Amyloid-β challenge: Apply Aβ oligomers (1-500 nM) to slices and assess PTK2B-dependent effects on synaptic plasticity

• Molecular interaction studies:

  • Co-immunoprecipitation: Use validated IP protocol (0.5-4.0 μg antibody for 1.0-3.0 mg lysate)

  • Key interactions: Focus on PTK2B interactions with Fyn kinase, which shows direct cross-activation

  • Additional targets: PrPC, mGluR5, NMDA receptor subunits

  • Phosphorylation analysis: Monitor phosphorylation of NMDAR subunits, especially pNR2B Y1472

• Dendritic spine analysis:

  • Visualization: Use DiI labeling or GFP expression in neurons

  • Parameters: Measure spine density, morphology (stubby, mushroom, thin), and dynamic changes

  • Perturbation: Compare wild-type vs. PTK2B knockout/knockdown neurons, with and without Aβ challenge

• In vivo models:

  • PTK2B knockout mice: Study baseline and AD-related phenotypes

  • AD mouse models: Evaluate PTK2B phosphorylation status and test PTK2B modulators

  • Behavioral testing: Focus on hippocampal-dependent memory tasks

This systematic approach allows for comprehensive investigation of PTK2B's role in neuronal function and AD pathology, as supported by findings in result .

How can I effectively study PTK2B's role in antiviral immunity using molecular and cellular approaches?

To investigate PTK2B's role in antiviral immunity, implement the following approaches based on validated methodologies:

• Cell-based viral infection models:

  • Cell types: Mouse embryonic fibroblasts (MEFs), macrophages (RAW 264.7), dendritic cells, and human macrophage THP1 cells

  • Viral systems: HSV1-GFP (DNA virus) and VSVΔM51-GFP (RNA virus)

  • Alternative stimuli: HT-DNA (human telomeric DNA) and poly(I:C) (synthetic dsRNA)

• Gene expression analysis:

  • Key antiviral genes: Monitor IFNB1, IFIT1, and CXCL10 using qRT-PCR

  • Expected effect: PTK2B knockdown/knockout significantly reduces expression of these genes upon viral challenge

  • Time course: Measure expression at 6-24 hours post-infection

• Protein phosphorylation analysis:

  • Key signaling components: Monitor phosphorylation of STING, TBK1 (particularly at Y591), and IRF3

  • Detection method: Western blotting with phospho-specific antibodies

  • Controls: Include total protein antibodies and loading controls

  • Expected result: PTK2B depletion reduces phosphorylation of these components

• Genetic manipulation approaches:

  • Knockdown: Use lentivirus-mediated shRNA or antisense oligonucleotides (ASOs)

  • Knockout: Generate using CRISPR-Cas9 system as described in result

  • Overexpression: Transfect PTK2B expression constructs to enhance antiviral signaling

  • Mutational analysis: Generate Y591F TBK1 mutant to confirm phosphorylation site importance

• Protein-protein interaction studies:

  • Co-immunoprecipitation: Demonstrate PTK2B interaction with TBK1 and STING

  • Domain mapping: Define interaction regions between PTK2B and TBK1/STING

  • Functional consequence: Demonstrate that PTK2B promotes TBK1 and STING oligomerization

• In vivo models:

  • PTK2B-deficient mice: Challenge with lethal HSV-1 or VSV infection

  • Expected outcome: Increased susceptibility to viral infection compared to control mice

  • Measurement: Viral titers, survival rates, tissue pathology, and inflammatory markers

These approaches provide a comprehensive experimental framework for investigating PTK2B's role in antiviral immunity, based on validated methods described in result .

What are the most common technical challenges when using PTK2B antibodies and how can they be resolved?

Additional tissue-specific considerations:

• Brain tissue: Requires thorough perfusion (in vivo) or rapid fixation (post-mortem) to preserve PTK2B phosphorylation status

• Immune cells: Works well with PTK2B antibodies in Jurkat cells, which express high levels of the protein

• Cell lines vs. primary tissues: Antibody performance may vary; validate in your specific system

How can I optimize double-labeling immunofluorescence protocols to study PTK2B co-localization with other proteins?

Optimizing double-labeling immunofluorescence for PTK2B co-localization studies requires careful consideration of antibody compatibility and protocol modifications:

• Antibody selection and validation:

  • PTK2B primary antibody: Select antibodies raised in different host species than your co-staining target (e.g., mouse anti-PTK2B with rabbit anti-target protein)

  • Validated pairs: For neuronal studies, mouse anti-PTK2B (67141-1-Ig) pairs well with rabbit antibodies against PSD-95, NMDAR subunits, or Fyn

  • Phospho-specific options: For phosphorylation studies, rabbit anti-pPyk2 Y402 (Abcam #ab131543) has been validated for IF in neuronal tissues

• Protocol optimization:

  • Sequential vs. simultaneous incubation:

    • Sequential: Apply first primary antibody, complete detection, then apply second primary (minimizes cross-reactivity)

    • Simultaneous: Mix compatible primaries at optimal dilutions (faster but requires thorough validation)

  • Antigen retrieval: TE buffer pH 9.0 works optimally for PTK2B detection in tissues

  • Blocking: Extended blocking (2h) with 10% normal serum from the species of both secondary antibodies

  • Controls: Single-labeled controls for each antibody to assess bleed-through

• Signal detection optimization:

  • Fluorophore selection: Choose spectrally distinct fluorophores (e.g., Alexa 488/FITC for PTK2B + Alexa 594/Texas Red for target)

  • Signal amplification: Consider tyramide signal amplification for low-abundance targets

  • Sequential imaging: For closely overlapping emission spectra, use sequential scanning

  • Standardized exposure: Use identical exposure settings for experimental and control samples

• Co-localization analysis:

  • Qualitative assessment: Yellow/orange in merged images indicates potential co-localization

  • Quantitative metrics: Calculate Pearson's or Manders' coefficients using ImageJ/Fiji plugins

  • Advanced analysis: Use line scan profiles across subcellular structures or super-resolution techniques for precise localization

• Application-specific optimizations:

  • Neuronal studies: Focus on dendritic spines and synapses where PTK2B regulates function

  • Immune cells: Examine co-localization with TBK1 and STING in the context of antiviral signaling

  • Fixed vs. live imaging: Consider fluorescently-tagged PTK2B constructs for dynamic studies

These optimizations will help ensure reliable co-localization results when studying PTK2B interactions with functionally relevant partners.

How does PTK2B contribute to inflammatory disease mechanisms based on recent research?

Recent investigations have revealed PTK2B's multifaceted role in inflammatory disease mechanisms, particularly in ulcerative colitis (UC):

• Expression patterns in inflammatory conditions:

  • PTK2B expression is significantly elevated in inflamed mucosa from UC patients compared to healthy controls

  • Expression levels positively correlate with disease severity

  • PTK2B is expressed in neutrophils and regulated by inflammatory cytokines

• Regulation of neutrophil function:

  • PTK2B modulates key neutrophil inflammatory functions:

    • Reactive oxygen species (ROS) production

    • Myeloperoxidase (MPO) release

    • Antimicrobial peptide (S100a8 and S100a9) generation

  • Pharmacological inhibition of PTK2B with TAE226 markedly reduces these inflammatory mediators

• Cellular migration regulation:

  • PTK2B enhances neutrophil migration by regulating CXCR2 and GRK2 expression

  • This regulation occurs via the p38 MAPK pathway

  • This migration enhancement may be protective in intestinal inflammation contexts

• Paradoxical protective role in colitis:

  • Despite promoting neutrophil inflammatory functions, PTK2B knockout mice display more severe colitis symptoms in DSS-induced models

  • This suggests PTK2B plays a complex role in balancing inflammation and tissue repair

  • The protective effect may be due to proper neutrophil recruitment and function at inflammatory sites

• Regulation by TNF-α signaling:

  • TNF-α promotes PTK2B expression in neutrophils

  • UC patients treated with infliximab (anti-TNF-α) show significantly reduced PTK2B levels

  • This positions PTK2B as a downstream effector in TNF-α-mediated inflammatory pathways

These findings collectively suggest that PTK2B functions as a context-dependent regulator of inflammation, promoting neutrophil migration while potentially limiting excessive tissue damage in inflammatory bowel disease .

What are the latest findings regarding PTK2B's role in Alzheimer's disease pathology?

Recent research has identified PTK2B (Pyk2) as a significant risk factor for late-onset Alzheimer's disease (AD) with specific neuronal mechanisms:

• Neuronal localization and function:

  • PTK2B localizes specifically to neurons in adult brain

  • It plays a critical role in synaptic plasticity regulation

  • While not significantly involved in synapse formation or basal synaptic transmission, PTK2B regulates activity-dependent plasticity

• Synaptic plasticity modulation:

  • PTK2B deletion specifically suppresses long-term depression (LTD) induction in the hippocampal Schaffer collateral pathway

  • LTD dysregulation is increasingly recognized as an early mechanism in AD pathogenesis

  • PTK2B deletion does not alter LTP under normal conditions

• Amyloid-β response mechanisms:

  • PTK2B mediates amyloid-β oligomer (Aβo)-induced synaptic dysfunction

  • This aligns with previous findings that Aβo inhibition of LTP, enhancement of LTD, and damage to dendritic spines requires Fyn kinase

  • There is direct interaction and cross-activation between PTK2B and Fyn in these pathways

• Molecular interaction network:

  • PTK2B interacts with several AD-relevant proteins:

    • Direct interaction with Fyn kinase

    • Involvement in the PrPC-mGluR5-Fyn pathway implicated in AD

    • Regulation of NMDA receptor functions through phosphorylation events

• Therapeutic implications:

  • AD transgenic mice exhibit elevated PTK2B function

  • This elevation can be normalized by:

    • PrPC deletion

    • mGluR5 deletion or inhibition

    • Fyn inhibition

  • Normalization parallels rescue of synapses and memory, suggesting therapeutic potential

These findings collectively establish PTK2B as an important molecular player in AD pathogenesis, particularly through its role in synaptic plasticity dysregulation and amyloid-β response pathways, positioning it as a potential therapeutic target .

How does PTK2B regulate antiviral immune responses at the molecular level?

Recent research has revealed PTK2B as a critical regulator of antiviral immunity through specific molecular mechanisms:

• Direct modulation of key signaling components:

  • PTK2B directly phosphorylates TBK1 at residue Tyr591

  • This phosphorylation event significantly increases TBK1 oligomerization and activation

  • PTK2B also interacts with STING and promotes its oligomerization through a kinase-independent mechanism

  • These dual mechanisms enhance STING-TBK1 activation, a central axis in antiviral responses

• Regulation of antiviral gene expression:

  • PTK2B depletion significantly reduces transcription of key antiviral genes:

    • Type I interferons (IFNB1)

    • Interferon-stimulated genes (IFIT1)

    • Chemokines (CXCL10)

  • This effect is observed with both DNA virus (HSV-1) and RNA virus (VSV) infections

  • Similar effects are seen with synthetic nucleic acid stimuli (HT-DNA, poly(I:C))

• Cell-type specific functions:

  • PTK2B regulates antiviral signaling in multiple cell types:

    • Mouse embryonic fibroblasts (MEFs)

    • Macrophages (THP1, RAW 264.7)

    • Dendritic cells

  • Genetic experiments demonstrate conserved function across these cell types

• Pathway-specific interactions:

  • Co-immunoprecipitation experiments show PTK2B interaction with:

    • TBK1 (but not RIG-I or MAVS) during RNA virus infection

    • STING during DNA virus infection

  • Domain mapping experiments identify specific interaction regions between these proteins

• In vivo significance:

  • Ptk2b-knockout mice show increased susceptibility to lethal:

    • Herpes simplex virus type 1 (HSV-1) infection

    • Vesicular stomatitis virus (VSV) infection

  • This demonstrates the physiological importance of PTK2B in antiviral defense

This molecular understanding of PTK2B's role in antiviral immunity positions it as a potential therapeutic target for enhancing antiviral responses or treating viral infection-associated immunopathology .

What are the optimal protocols for studying PTK2B's role in cell migration and adhesion dynamics?

To effectively study PTK2B's role in cell migration and adhesion, implement these methodologically robust approaches:

• Live cell imaging of migration:

  • Setup: Use phase contrast or fluorescence microscopy with environmental chamber (37°C, 5% CO₂)

  • Cell labeling: Express fluorescent PTK2B fusion constructs or use CellTracker dyes

  • Acquisition parameters: Capture images every 5-10 minutes for 12-24 hours

  • Analysis: Track individual cells using ImageJ/Fiji with Manual Tracking or TrackMate plugins

  • Key parameters: Measure velocity, directionality, persistence, and track length

• Scratch wound healing assay:

  • Cell preparation: Culture cells to confluence in multiwell plates

  • Wounding: Create consistent scratches using pipette tips or wound-making tools

  • PTK2B manipulation: Compare control vs. PTK2B-knockdown/knockout cells or use PTK2B inhibitor TAE226

  • Documentation: Capture images at 0, 6, 12, and 24 hours

  • Quantification: Measure wound closure percentage using ImageJ

• Transwell migration assay:

  • Optimal for: Neutrophils, macrophages, cancer cells

  • Chemoattractants: Cell-type specific (fMLP for neutrophils, SDF-1 for cancer cells)

  • PTK2B role assessment: Compare migration with/without PTK2B inhibition

  • Analysis: Count migrated cells after 4-24 hours (cell-type dependent)

  • Expected result: PTK2B enhances neutrophil migration by regulating CXCR2 and GRK2 expression via p38 MAPK pathway

• Focal adhesion dynamics:

  • Markers: Express fluorescent paxillin, vinculin, or talin along with PTK2B

  • Imaging: Use total internal reflection fluorescence (TIRF) microscopy

  • Time intervals: Capture images every 1-2 minutes for 1-2 hours

  • Analysis: Measure focal adhesion assembly/disassembly rates and lifetime

  • PTK2B manipulation: Compare wild-type vs. phospho-mutants of PTK2B

• Adhesion strength assays:

  • Centrifugal assay: Seed cells on protein-coated plates, invert and centrifuge

  • Shear flow assay: Apply defined fluid shear stress to adherent cells

  • Quantification: Count remaining adherent cells after challenge

  • PTK2B specificity: Compare with FAK inhibition to distinguish functions

• Signaling pathway analysis:

  • Key phosphorylation sites: Monitor PTK2B Y402 and Y579 phosphorylation

  • Downstream effectors: Evaluate p38 MAPK, Src family kinases, and small GTPases

  • Inhibitor approach: Use pathway-specific inhibitors to define hierarchy

  • Time course: Assess signaling events from early (minutes) to late (hours)

These methodologies provide a comprehensive experimental framework for investigating PTK2B's functions in cell migration and adhesion dynamics, particularly relevant to inflammatory and cancer research contexts.

What are the critical considerations when generating and validating PTK2B knockout or knockdown models?

Generating and validating PTK2B knockout or knockdown models requires careful attention to the following methodological considerations:

• Selection of targeting strategy:

  • CRISPR-Cas9 knockout:

    • Design multiple gRNAs targeting early exons (validated approach from result )

    • Consider potential off-target effects using prediction tools

    • For neuronal studies, be aware that complete PTK2B knockout affects LTD but not basal synaptic parameters

  • shRNA knockdown:

    • Design 3-4 independent shRNAs targeting different regions

    • Validated target sequences from result : use lentivirus-mediated shRNA in THP1 or RAW 264.7 cells

    • Include non-targeting control shRNA with similar GC content

  • Antisense oligonucleotides (ASOs):

    • Alternative approach validated in result

    • Particularly useful for in vivo or primary cell applications

    • Provides temporal control of knockdown

• Validation of knockout/knockdown efficiency:

  • Protein level validation (essential):

    • Western blot using validated antibodies (67141-1-Ig or 17592-1-AP)

    • Expected 80-95% reduction for effective knockdown

    • Complete absence of bands at 112-115 kDa for knockout

  • Genomic validation (for CRISPR models):

    • PCR and sequencing of target region

    • Analysis of indel patterns and frameshift mutations

    • Confirmation of biallelic targeting

  • mRNA level validation (complementary):

    • qRT-PCR with primers spanning multiple exons

    • Analysis of potential truncated transcripts

• Functional validation:

  • Signal transduction:

    • Assess phosphorylation of known downstream targets (TBK1, IRF3)

    • Challenge with stimuli known to activate PTK2B (viral infection, TNF-α)

  • Cellular phenotypes:

    • For immune cells: Evaluate antiviral gene expression (IFNB1, IFIT1, CXCL10)

    • For neutrophils: Assess migration capacity and ROS production

    • For neurons: Examine synaptic plasticity (LTD specifically)

• Control for compensatory mechanisms:

  • Related protein expression: Monitor FAK (PTK2) expression which may compensate

  • Inducible systems: Consider doxycycline-inducible knockdown to minimize adaptation

  • Acute vs. chronic depletion: Compare acute inhibition to genetic knockout

• Rescue experiments (critical for specificity):

  • Re-expression of wild-type PTK2B: Should restore normal phenotype

  • Mutant variants: Test phosphorylation site mutants (Y402F, Y579F) for functional significance

  • Expression level control: Match endogenous expression levels to avoid overexpression artifacts

• In vivo models considerations:

  • Viability: PTK2B knockout mice are viable but show specific phenotypes

  • Tissue-specific knockouts: Consider for studying tissue-specific functions

  • Phenotypic assessment: Evaluate relevant disease models (viral infection susceptibility, colitis severity)