PTPRM Antibody

What is PTPRM and what cellular functions does it regulate?

PTPRM (also known as PTPμ, PTPRμ, and RPTPμ) is a receptor-type protein tyrosine phosphatase that mediates homotypic cell-cell interactions. It functions by dephosphorylating tyrosine residues on target proteins . PTPRM promotes CTNND1 (p120 catenin) dephosphorylation and prevents its cytoplasmic localization, directing SLC2A4 to the plasma membrane for glucose transport function . This phosphatase is highly expressed in pulmonary vascular epithelia, where its interactions with cadherins are important in regulating barrier permeability . Structurally, PTPRM contains MAM and Fibronectin III domains on its extracellular side that mediate intercellular binding, affecting adhesion and contact inhibition .

What applications are PTPRM antibodies validated for in research settings?

Based on the search results, PTPRM antibodies have been validated for multiple applications:

• Western blot (WB) with specific bands detected at approximately 280 kDa

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Immunofluorescence (IF)

• ELISA with dilutions ranging from 1:2000 to 1:10000

When selecting a PTPRM antibody, researchers should verify that it has been validated for their specific application and target species, as reactivity may vary between human, mouse, and rat samples .

How should PTPRM antibodies be stored to maintain optimal activity?

For maximum stability and activity retention, PTPRM antibodies require specific storage conditions:

• Unopened/lyophilized antibodies: Store at -20°C to -70°C for up to 12 months from receipt date

• After reconstitution: Store at 2-8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution: Store at -20°C to -70°C for up to 6 months

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Some PTPRM antibodies are supplied in storage buffer containing pH 7.4 PBS, 0.05% NaN₃, and 40% glycerol

Proper storage is critical as repeated freeze-thaw cycles can significantly degrade antibody performance and specificity.

How can researchers validate the specificity of PTPRM antibodies?

To ensure experimental rigor when working with PTPRM antibodies, researchers should implement multiple validation strategies:

• Multiple antibody approach: Use antibodies targeting different PTPRM epitopes and compare results (e.g., antibodies targeting Glu21-Lys742 vs. aa 1150-1450 )

• Appropriate controls:

  • Positive controls: Cell lines known to express PTPRM (HepG2, CHP-100 human neuroblastoma cells)

  • Negative controls: PTPRM knockdown cells using ribozyme transgenes

  • Tissue controls: Normal small intestine enterochromaffin cells that naturally express PTPRM

• Complementary techniques: Confirm protein detection with mRNA expression analysis by quantitative RT-PCR

• IHC validation controls:

  • Omit primary antibody (should show no staining)

  • Include synaptophysin staining as tissue quality control in neuroendocrine tissues

  • Use isotype control antibodies to rule out non-specific binding

What considerations are important when analyzing PTPRM expression in tumor samples?

When studying PTPRM in tumors, researchers must address several complex issues:

• Heterogeneous expression patterns: Small intestinal neuroendocrine tumors (SI-NETs) often show variable heterogeneous staining with both negative and positive areas within the same sample . This requires careful assessment of multiple tumor regions.

• Subcellular localization variations: PTPRM staining may be exclusively cytoplasmic or show combined cytoplasmic/nuclear patterns in tumors . Since the expected localization is primarily membranous/cytoplasmic, nuclear staining may indicate altered function.

• Primary vs. metastatic expression: Significant differences in PTPRM expression between primary tumors and their metastases have been reported, with metastases typically showing lower expression .

• Epigenetic regulation: PTPRM can be epigenetically silenced in tumors, affecting detection. Treatment with DNA methylation inhibitors like 5-aza-2′-deoxycytidine can dramatically increase PTPRM expression (>100-fold in CNDT2.5 cells) , suggesting epigenetic regulation should be considered when interpreting negative results.

How do you interpret contradictory findings regarding PTPRM's role in different cancer types?

The literature reveals context-dependent functions of PTPRM across cancer types:

To reconcile these contradictions:

• Context-dependent hypothesis: PTPRM may function differently depending on tissue type and molecular context

• Experimental approach:

  • Perform both gain- and loss-of-function studies in your specific model

  • Assess impact on hallmark cancer processes (proliferation, migration, invasion)

  • Analyze downstream signaling pathways affected by PTPRM manipulation

• Substrate analysis: Identify and validate PTPRM substrates in your specific cancer model, as different substrates may explain different outcomes

• Correlation with clinical features: For example, in cervical cancer, high PTPRM expression correlates with tumor size >4cm (p=0.019)

What methods are recommended for studying PTPRM's role in cell adhesion and EMT?

PTPRM regulates both cell-cell adhesion and epithelial-mesenchymal transition (EMT), requiring specific experimental approaches:

• Cell density experiments: Culturing cells to high density concentrates PTPRM at sites of tight contact and induces proteolytic cleavage of its extracellular domain . Monitor PTPRM localization and processing at different cell densities.

• Cytoskeletal visualization: PTPRM knockdown in SiHa cells changed their morphology from elongated to rounded, as visualized by TRITC phalloidin staining .

• EMT marker analysis: After PTPRM knockdown, monitor changes in:

  • Epithelial markers: E-cadherin (increased after PTPRM knockdown)

  • Mesenchymal markers: N-cadherin, Vimentin (decreased after PTPRM knockdown)

  • EMT transcription factors: Snail (decreased after PTPRM knockdown)

• VEGF-C regulation: PTPRM knockdown decreased VEGF-C expression at both mRNA and protein levels in cervical cancer cells, suggesting a role in lymphangiogenesis .

What strategies can enhance detection of low-abundance or downregulated PTPRM?

When PTPRM is downregulated in tissues, several approaches can enhance detection:

• Signal amplification methods:

  • Use tyramide signal amplification for IHC/IF

  • Consider high-sensitivity chemiluminescent substrates for Western blot with extended exposure times

• Sample enrichment:

  • Perform immunoprecipitation to concentrate PTPRM before detection

• Epigenetic modification:

  • PTPRM can be epigenetically silenced in cancer

  • Treatment with DNA methylation inhibitor 5-aza-2′-deoxycytidine increased PTPRM expression >100-fold in CNDT2.5 cells

  • S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) induced PTPRM expression approximately 10-fold in CNDT2.5 cells

• Complementary approaches:

  • Combine antibody-based detection with qRT-PCR to confirm expression patterns at both protein and mRNA levels

How can researchers optimize Western blot protocols for PTPRM detection?

For successful Western blot detection of PTPRM:

• Sample preparation:

  • Use cell lines with known PTPRM expression as positive controls (HepG2 human hepatocellular carcinoma or CHP-100 human neuroblastoma cell lines)

• Technical parameters:

  • Use PVDF membrane for optimal protein transfer and binding

  • Probe with 1-2 μg/mL of anti-PTPRM antibody

  • Use HRP-conjugated secondary antibodies for detection

  • Expect a band at approximately 280 kDa under reducing conditions

  • Consider using Immunoblot Buffer Group 1 as described in the literature

• Protein size considerations:

  • Full-length PTPRM is approximately 280 kDa

  • Be aware that proteolytic processing may generate additional fragments

What controls are essential when studying PTPRM in knockdown or knockout models?

When using PTPRM genetic manipulation models:

• Validation of knockdown efficiency:

  • Verify using multiple methods: RT-PCR, real-time quantitative PCR, and Western blot

  • Example: The knockdown of PTPRM in MDA-MB-231 and MCF-7 cells was verified using these complementary approaches

• Appropriate vector controls:

  • Include empty plasmid controls alongside ribozyme transgenes

  • Use matched parental cell lines as additional controls

• Functional validation:

  • Monitor changes in:

    • EMT markers (E-cadherin, Snail, N-cadherin, Vimentin)

    • Cytoskeletal organization using phalloidin staining

    • Cell proliferation and apoptosis rates

• Multiple cell lines:

  • Test effects in different cellular contexts (e.g., both ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells)

How can researchers investigate the phosphatase activity of PTPRM?

To study PTPRM's enzymatic function:

• Phosphatase-dead mutants as controls:

  • C1095S and C1095S/C1389S mutants can serve as catalytically inactive controls

  • These can be generated using site-directed mutagenesis as described in the literature

• Substrate phosphorylation analysis:

  • Monitor p120 catenin (CTNND1) phosphorylation status, which is regulated by PTPRM

  • Use phospho-tyrosine-specific antibodies to detect changes in substrate phosphorylation

• Signaling pathway analysis:

  • PTPRM influences the ERK1/2 signaling pathway in psoriatic skin

  • Two genes, PTPRM and NELL2, affect the ERK1/2 pathway and their deregulation supports excessive ERK1/2 activation in psoriasis

• Functional readouts:

  • Assess changes in:

    • Cell migration and invasion capacity

    • Cell-cell adhesion strength

    • Colony formation ability (PTPRM negatively regulates cell growth and colony formation)

What approaches are recommended for studying PTPRM in disease-specific contexts?

For disease-specific research:

• Psoriasis models:

  • Use tissue-engineered two-layered (dermis and epidermis) human skin substitutes enriched in T cells

  • Monitor PTPRM expression in relation to ERK1/2 pathway activation

  • Consider combining with RSK inhibition (a downstream effector of ERK1/2)

• Cancer models:

  • For colorectal cancer: Use matching sets of colon mucosa-adenoma-carcinoma samples to track PTPRM changes during disease progression

  • For neuroendocrine tumors: Compare PTPRM expression between primary tumors and metastases

  • For cervical cancer: Correlate PTPRM expression with clinicopathological features using the following parameters :

• Epigenetic regulation:

  • Investigate promoter methylation status using pyrosequencing

  • Test effects of epigenetic modifiers (5-aza-2′-deoxycytidine, DZNep)

  • Correlate with expression levels to establish epigenetic regulation mechanisms

What emerging areas of PTPRM research show promise for therapeutic development?

Several promising research directions emerge from the current literature:

• Targeting PTPRM in cancers where it promotes progression:

  • In cervical cancer, PTPRM promotes tumor growth and lymph node metastasis

  • Developing inhibitors or neutralizing antibodies could have therapeutic potential

• Restoring PTPRM expression where it acts as a tumor suppressor:

  • Epigenetic modifiers (DNA methylation inhibitors) dramatically increase PTPRM expression in some cancer models

  • This approach could be valuable in colorectal cancer and SI-NETs where PTPRM shows tumor suppressive properties

• Psoriasis treatment approaches:

  • Inhibition of RSK (a downstream effector of ERK1/2) showed therapeutic potential in psoriatic skin models where PTPRM is deregulated

  • This suggests targeting signaling pathways affected by PTPRM dysregulation rather than PTPRM itself

• Cell adhesion and barrier function applications:

  • PTPRM's role in regulating pulmonary vascular epithelia barrier permeability suggests applications in understanding and treating conditions involving endothelial barrier dysfunction

How can researchers better address the context-dependent functions of PTPRM?

To resolve the seemingly contradictory roles of PTPRM:

• Comprehensive tissue profiling:

  • Compare PTPRM expression, localization, and associated signaling networks across multiple tissue types

  • Identify tissue-specific binding partners that might explain differential functions

• Multi-omics approach:

  • Combine proteomics, phosphoproteomics, and transcriptomics to develop comprehensive models of PTPRM function

  • Identify context-specific substrates that might explain different outcomes

• In vivo modeling:

  • Develop tissue-specific conditional knockout models to evaluate PTPRM function in specific physiological contexts

  • Use advanced disease models like organoids and patient-derived xenografts

• Structural biology approaches:

  • Investigate how structural differences in PTPRM between tissues might affect its function and substrate specificity

  • Develop structure-based screening for tissue-specific inhibitors or activators