PTPN7 Antibody

What is PTPN7 and what are its key structural features?

PTPN7, also known as hematopoietic protein tyrosine phosphatase (HePTP), is a 38-kDa cytoplasmic protein tyrosine phosphatase consisting of a C-terminal catalytic domain and a short N-terminal extension containing the kinase interaction motif. It was originally cloned from human T lymphocytes and is expressed primarily in cells of hematopoietic lineage, including neutrophils, megakaryocytes, erythrocytes, and lymphocytes . The protein contains a conserved 16 amino acid sequence at its N-terminus that is characteristic of the R7 family of protein tyrosine phosphatases .

What are the primary cellular functions of PTPN7?

PTPN7 functions primarily as a negative regulator of mitogen-activated protein kinases (MAPKs), particularly ERK1/2 and p38. In T cells, PTPN7 dephosphorylates ERK and thereby negatively regulates T cell activation . In platelets, PTPN7 regulates ERK1/2, which controls thromboxane A2 (TXA2) generation and subsequent platelet functional responses . Additionally, PTPN7 plays a regulatory role in macrophages by inhibiting LPS-stimulated production of TNF-α . PTPN7 is the only known PTP whose expression in T-lymphocytes is induced by IL-2 .

What is the expression pattern of PTPN7 in normal and disease tissues?

PTPN7 is primarily expressed in cells of hematopoietic lineage. In cancer, PTPN7 expression is upregulated in several tumor types compared to para-tumor specimens, including STAD, CHOL, HNSC, ESCA, BRCA, KIPR, KIRC, LUAD, LIHC, CESE, and GMB . In breast cancer specifically, PTPN7 expression is higher in tumor tissues compared to paired para-tumor tissues .

What criteria should be considered when selecting a PTPN7 antibody for research?

When selecting a PTPN7 antibody, researchers should consider:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, ELISA, etc.)

• Species reactivity: Ensure the antibody recognizes PTPN7 in your species of interest

• Epitope recognition: Consider whether the antibody targets the N-terminal or C-terminal region

• Validation data: Review published literature citing the antibody

• Clone type: Determine whether monoclonal or polyclonal is more suitable for your application

Available PTPN7 antibodies have demonstrated reactivity with human, mouse, and rat samples .

What validation methods are recommended for PTPN7 antibodies?

To validate PTPN7 antibodies, researchers should:

• Use PTPN7 knockout samples as negative controls (as demonstrated in PTPN7 KO mice studies )

• Perform Western blot analysis to confirm the expected molecular weight (38-40 kDa)

• Compare staining patterns in tissues known to express PTPN7 (lymphoid tissues) versus those with minimal expression

• Use recombinant PTPN7 protein as a positive control

• Compare results with multiple PTPN7 antibodies targeting different epitopes

• Validate expression patterns with orthogonal methods (e.g., qRT-PCR for mRNA expression)

What are the recommended protocols for PTPN7 detection by Western blot?

For optimal Western blot detection of PTPN7:

• Prepare cell/tissue lysates in standard RIPA buffer with protease inhibitors

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk or BSA in TBST

• Incubate with primary PTPN7 antibody at dilutions of 1:500-1:3000

• Detect PTPN7 at the expected molecular weight of 38-40 kDa

• Include positive controls (Raji cells, K-562 cells) and negative controls (PTPN7 knockout samples if available)

What are the optimal conditions for immunohistochemical detection of PTPN7?

For IHC detection of PTPN7:

• Fix tissues in 10% neutral-buffered formalin and embed in paraffin

• Section tissues at 4-5 μm thickness

• Perform antigen retrieval with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0 may be used)

• Block endogenous peroxidase and non-specific binding

• Incubate with PTPN7 antibody at 1:200-1:800 dilution

• Use appropriate detection systems (e.g., DAB and hematoxylin counterstain)

• Include positive control tissues (human tonsillitis tissue, human lymphoma tissue)

• Evaluate staining using semi-quantitative immunoreactivity score (IRS) on a 12-point scale

How can researchers accurately measure PTPN7 phosphatase activity?

To measure PTPN7 phosphatase activity:

• Immunoprecipitate endogenous PTPN7 from cell lysates using anti-PTPN7 antibodies

• Assess phosphatase activity using synthetic phosphopeptides or phosphorylated proteins (particularly ERK and p38 MAPK) as substrates

• Measure the release of inorganic phosphate using malachite green assay or other phosphate detection methods

• Include controls such as phosphatase inhibitors (sodium orthovanadate) and heat-inactivated samples

• Compare activity between experimental conditions (e.g., with/without LPS stimulation)

Research has shown that PTPN7 phosphatase activity decreases in proportion to the decrease of PTPN7 expression following LPS treatment in RAW 264.7 cells .

What methods can be used to study PTPN7's role in MAPK signaling pathways?

To investigate PTPN7's role in MAPK signaling:

• Overexpress wild-type or mutant PTPN7 (e.g., using FLAG-tagged PTPN7 constructs)

• Knockdown PTPN7 using siRNA or CRISPR-Cas9

• Measure phosphorylation levels of ERK1/2 and p38 MAPK by Western blot

• Examine downstream effects (e.g., TXA2 generation, cytokine production)

• Use specific inhibitors of MAPK pathways (U0126 for MEK/ERK, SB203580 for p38) to confirm involvement

• Perform co-immunoprecipitation studies to demonstrate physical interaction between PTPN7 and MAPKs

Studies have demonstrated that PTPN7 knockout mouse platelets exhibit increased ERK1/2 phosphorylation compared to wild-type, without affecting upstream MEK activation or p38 MAPK phosphorylation .

How can researchers investigate PTPN7's role in platelet function?

To study PTPN7 in platelet function:

• Isolate platelets from wild-type and PTPN7 knockout mice

• Perform platelet functional assays:

  • Aggregation in response to agonists (PAR4, ADP, GPVI)

  • Dense granule secretion (ATP release)

  • TXA2 generation (measured by TXB2 assay)

  • Flow cytometry for p-selectin expression and active GPIIb/IIIa

• Pre-treat platelets with inhibitors (e.g., indomethacin for cyclooxygenase)

• Analyze signaling pathways by measuring phosphorylation of ERK1/2, MEK, and p38 MAPK

Research has shown that PTPN7 KO mouse platelets exhibit increased platelet functional responses, including aggregation, dense granule secretion, and TXA2 generation, compared to wild-type platelets .

What blood parameters should be evaluated in PTPN7 knockout models?

When using PTPN7 knockout models, researchers should evaluate:

Blood cell counts in PTPN7 KO mice do not significantly differ from wild-type littermates, indicating that PTPN7 deletion does not alter hematopoiesis .

How can researchers evaluate PTPN7's correlation with immune cell infiltration in tumors?

To assess PTPN7's relationship with immune infiltration:

• Analyze gene expression data from public databases (e.g., TCGA)

• Use computational tools like TIMER to estimate abundance of tumor-infiltrating immune cells

• Evaluate correlation between PTPN7 expression and markers of immune cells (B cells, CD8+ T cells, CD4+ T cells, neutrophils, macrophages, dendritic cells)

• Assess tumor purity in relation to PTPN7 expression

• Perform immunohistochemical co-staining of tumor samples for PTPN7 and immune cell markers

• Analyze correlation between PTPN7 and immune checkpoint molecules (PD-L1, CTLA-4)

Studies have shown that PTPN7 expression is negatively correlated with tumor purity but positively correlated with multiple immune cell infiltration in most cancer types .

What methodologies are appropriate for investigating PTPN7 as a biomarker for immunotherapy response?

To investigate PTPN7 as an immunotherapy biomarker:

• Analyze RNA-seq data from patients receiving immunotherapy (e.g., from GEO datasets)

• Compare PTPN7 expression between responders and non-responders

• Assess correlation between PTPN7 and established biomarkers (PD-L1, TMB)

• Perform ROC analysis to determine diagnostic performance

• Evaluate PTPN7 expression before and after treatment

• Combine PTPN7 with other markers to develop predictive signatures

• Validate findings in independent cohorts

Research has shown that PTPN7 is upregulated in patients with better immunotherapeutic response in multiple cancer types, and its predictive value has been demonstrated in independent GEO cohorts .

What methods can be used to analyze PTPN7 gene expression dynamics?

For analyzing PTPN7 gene expression dynamics:

• Quantitative RT-PCR using specific primers:

  • PTPN7 mRNA (forward: 5′-CAGAGACAGCTGCCAACTCCGG-3′; reverse: 5′-CCATGAGCTGGTGGAGGGTCAG-3′)

  • Normalize to housekeeping genes (e.g., GAPDH)

• RNA-seq analysis to examine transcriptomic changes

• Time-course studies to capture dynamic changes (e.g., after LPS stimulation)

• Single-cell RNA-seq to examine cell-type-specific expression

• Analysis of transcription factor binding to PTPN7 promoter

• Investigation of epigenetic modifications affecting PTPN7 expression

Studies have shown that PTPN7 mRNA expression is suppressed within 2 hours of LPS treatment and returns to near basal levels by 24 hours .

What are the recommended controls for PTPN7 knockdown experiments?

When performing PTPN7 knockdown studies:

• Use multiple siRNA sequences targeting different regions of PTPN7 mRNA to ensure specificity:

  • siRNA #1: 5′-GAU CUA UCU CAG GGA UGA A (dTdT)-3′

  • siRNA #2: 5′-CAG GAU AGG CUU CUA AAG U (dTdT)-3′

• Include non-silencing negative control siRNAs

• Verify knockdown efficiency by Western blot and qRT-PCR

• Consider rescue experiments with siRNA-resistant PTPN7 constructs

• Use PTPN7 knockout cells/tissues as positive controls for knockdown

• Examine effects on known PTPN7 substrates (ERK1/2, p38 MAPK)

How can researchers address sensitivity issues when detecting endogenous PTPN7?

To enhance detection of endogenous PTPN7:

• Optimize cell lysis conditions (use phosphatase inhibitors to prevent autodephosphorylation)

• Enrich PTPN7 by immunoprecipitation before Western blot analysis

• Use cells known to express high levels of PTPN7 (lymphoid cell lines like Raji and K-562) as positive controls

• Optimize antibody concentration and incubation conditions

• Consider alternative detection methods (chemiluminescence vs. fluorescence)

• Use signal enhancement systems for IHC applications

• For low abundance samples, consider more sensitive techniques like digital PCR

What are the potential sources of data inconsistency in PTPN7 studies and how can they be addressed?

Potential sources of inconsistency and their solutions:

• Antibody specificity issues:

  • Validate antibodies using PTPN7 knockout samples

  • Use multiple antibodies targeting different epitopes

  • Include appropriate positive and negative controls

• Cell type-specific effects:

  • Clearly define cell types used in experiments

  • Consider tissue/cell-specific PTPN7 regulation

  • Use relevant primary cells when possible

• Experimental conditions:

  • Standardize stimulation protocols (concentration, timing)

  • Control for cell density and passage number

  • Ensure consistency in lysis and detection methods

• Data analysis:

  • Use appropriate normalization methods

  • Apply statistical tests suitable for the data distribution

  • Consider multiple reference genes for qRT-PCR

Research has shown intracellular content level of PTPN7 in breast cancer found in public databases was inconsistent, highlighting the importance of experimental validation .

What are the emerging applications of PTPN7 antibodies in understanding disease mechanisms?

Emerging applications include:

• Single-cell analysis of PTPN7 expression in tumor microenvironments

• Spatial transcriptomics to map PTPN7 expression within tissues

• Investigation of PTPN7 in immune checkpoint regulation

• Studies of PTPN7 in conjunction with transcription factor networks

• Exploration of PTPN7's role in resistance to targeted therapies

• Development of PTPN7 as a predictive biomarker for personalized immunotherapy

• Understanding PTPN7's function in non-hematopoietic tissues

What methodological advances could enhance PTPN7 functional studies?

Advanced methodologies for PTPN7 research include:

• CRISPR-Cas9-mediated generation of PTPN7 variants with specific phosphatase activity alterations

• Optogenetic approaches to temporally control PTPN7 activity

• Protein structural studies to develop specific PTPN7 inhibitors

• Multiplexed protein-protein interaction studies to map PTPN7 interactome

• Biosensors to monitor PTPN7 activity in live cells

• Multi-omics integration approaches to understand PTPN7's global effects

• Advanced animal models with tissue-specific or inducible PTPN7 knockout/overexpression