CTHRC1 Antibody

What is CTHRC1 and why is it significant in research applications?

CTHRC1 is a glycosylated, secreted protein that functions as a negative regulator of collagen matrix deposition. It is transiently expressed in the arterial wall in response to injury and contributes to vascular remodeling by inhibiting collagen expression and promoting cell migration . CTHRC1 is expressed in various cell types including renal epithelium, neurons, osteoblasts, and smooth muscle cells .

Research significance includes:

• Marker for differentiated smooth muscle in its unprocessed form

• Highly expressed in multiple human cancers (pancreatic, gastric, colon, lung)

• Associated with cancer progression and metastasis through regulation of tumor cell migration and adhesion

• Potential prognostic marker in gastric cancer

• Role in pancreatic stellate cells and fibroblast response to TGF-β1

What molecular forms of CTHRC1 exist and how do they affect antibody selection?

CTHRC1 exists in multiple molecular forms that researchers should consider when selecting antibodies:

• Full-length CTHRC1: 26-30 kDa (monomeric form)

• Dimeric form: approximately 52 kDa

• Trimeric form: approximately 78 kDa

• N-terminally truncated forms generated by proteolytic processing

In research settings, CTHRC1 can be detected as a mixture of monomer, dimer, and trimer forms, with respective molar percentages of approximately 25%, 27%, and 48% . The selection of antibodies should consider which form(s) you need to detect and whether the antibody recognizes the N-terminal region (propeptide) or other epitopes that may be masked in certain complexes .

What are the standard applications for CTHRC1 antibodies?

CTHRC1 antibodies have been validated for multiple research applications:

Each application requires optimization as sensitivity is sample-dependent .

How should I choose between monoclonal and polyclonal CTHRC1 antibodies?

The choice between monoclonal and polyclonal CTHRC1 antibodies depends on your experimental goals:

Monoclonal Antibodies (e.g., clone 5H1, 16D3, 16D4)

• Advantages: Highly specific for single epitopes, consistent lot-to-lot performance

• Best for: Detecting specific forms or domains of CTHRC1

• Example applications: When epitope specificity is critical, such as distinguishing processed vs. unprocessed forms

Polyclonal Antibodies

Research has demonstrated that domain-specific antibodies (such as antipro antibodies against the N-terminal peptide sequence) can localize full-length CTHRC1 in the cytoplasm of various tissues, while antibodies raised against the full-length protein may miss certain complexed forms .

What positive controls should I use to validate CTHRC1 antibody performance?

Based on published research, recommended positive controls include:

Recombinant Proteins:

• Purified CTHRC1 recombinant protein

• Conditioned media from CHO cells transduced with CTHRC1 adenovirus

Cell Lines:

• A375 cells (human melanoma)

• UACC-62 cells (melanoma)

• Jurkat cells (human T lymphocyte)

• HepG2 cells (human liver cancer)

• PAC1 cells (smooth muscle cells)

Tissue Samples:

• Mouse lung tissue

• Rat large intestine tissue

• Human kidney tissue

• Human placenta tissue

• Porcine aorta (for detection of complex forms)

The choice of positive control should be based on the specific form of CTHRC1 you aim to detect, as expression levels and forms vary across tissues and cell types.

How can CTHRC1 antibodies distinguish between intracellular and secreted forms?

Distinguishing between intracellular and secreted CTHRC1 requires careful antibody selection and experimental design:

Intracellular CTHRC1:

• Domain-specific antibodies against the N-terminus (antipro antibodies) can localize full-length CTHRC1 in the cytoplasm of vascular, gastrointestinal, and uterine smooth muscle cells as well as some neurons

• In differentiated smooth muscle, full-length CTHRC1 appears to form a complex with cytoplasmic proteins, making detection challenging with antibodies raised against aggregating forms

Secreted CTHRC1:

• Antibodies detecting the mature region can identify secreted forms in conditioned media

• When using CHO cells transduced with CTHRC1 adenovirus, secreted forms can be detected in the medium by immunoblotting with antibodies at concentrations of 50 ng/mL and below

For comprehensive analysis, researchers should consider:

• Collecting both cell lysates and conditioned media

• Using multiple antibodies recognizing different epitopes

• Comparing reducing and non-reducing conditions to identify complexes

How can I detect and analyze proteolytic processing of CTHRC1?

CTHRC1 undergoes proteolytic processing, generating various fragments with different molecular weights and potentially different functions. To study this process:

Experimental Approach:

• Plasmin Cleavage Analysis:

  • Incubate purified His-tagged CTHRC1 with human plasmin (0.05 U) at 37°C for 5-30 minutes

  • Use controls without plasmin for comparison

  • Resolve fragments by SDS-PAGE and transfer to PVDF membranes

  • Perform N-terminal peptide sequencing to identify cleavage sites

• Inhibition of Endogenous Plasmin:

  • Treat cells expressing CTHRC1 with 0.5 mmol/L ε-aminocaproic acid (EACA)

  • Collect conditioned media and cell lysates for immunoblotting

  • Compare fragment patterns with and without inhibitor

• Fragment Identification:

  • Use antibodies that recognize different epitopes to identify specific fragments

  • In PAC1 smooth muscle cells, several immunoreactive bands below 30 kDa can be observed (labeled as fragments b, c, d)

  • The majority of endogenous CTHRC1 in these cells is present as fragment c (approximately 20 kDa)

These approaches can help elucidate the biological significance of CTHRC1 processing in different cellular contexts.

What are the technical considerations for detecting CTHRC1 in cancer research applications?

CTHRC1 is highly expressed in multiple human cancers and plays significant roles in progression and metastasis. For cancer research applications, consider:

Tissue Selection and Processing:

• CTHRC1 overexpression has been documented in pancreatic, gastric, colon, hepatocellular, and lung cancers

• For prostate cancer, CTHRC1 expression has been correlated with tumor recurrence and may interact with PD-1/PD-L1 pathways

Antibody Selection:

• For tumor microenvironment studies, choose antibodies that can detect CTHRC1 in both tumor cells and stromal components

• Consider antibodies that specifically recognize secreted forms as these may be relevant for metastasis studies

Technical Approaches:

• Immunohistochemistry protocols should include antigen retrieval in 10 mmol/L citrate buffer for 15 minutes before staining for optimal results

• For comparative expression analysis between normal and tumor tissues, standardize tissue processing and staining conditions

• When studying CTHRC1's role in fibrotic processes in cancer, consider its interactions with the TGF-β pathway

Data Interpretation:

• Verify CTHRC1 localization (intracellular vs. extracellular) as this may provide insights into its functional state

• Consider CTHRC1 as a potential prognostic marker, particularly in gastric cancer

How can I address conflicting CTHRC1 antibody results across different applications?

When faced with conflicting results using CTHRC1 antibodies across different applications, consider these troubleshooting approaches:

Analysis of Protein Structure and Complexes:

• Under non-reducing conditions, CTHRC1 can form higher-order structures (monomers, dimers, trimers, tetramers)

• Under reducing conditions, most CTHRC1 runs as an apparent monomer

• In tissue samples (e.g., aorta), CTHRC1 can form high molecular weight complexes (>130 kDa) under non-reducing conditions

Epitope Accessibility Issues:

• Antibodies raised against different epitopes may give conflicting results due to epitope masking

• The N-terminal propeptide remains accessible in CTHRC1 complexes and can be detected by antipro antibodies, while other epitopes may be masked

• Some antibodies specifically recognize certain molecular weight forms

Experimental Design Solutions:

• Use multiple antibodies targeting different epitopes to cross-validate results

• Compare results under both reducing and non-reducing conditions

• Include appropriate positive controls (recombinant protein, cell lines known to express CTHRC1)

• Consider tissue-specific processing - CTHRC1 in brain primarily corresponds to fragment a, while fragment b is prevalent in lung tissue

• During wound healing in skin, CTHRC1 is processed into fragments c and d

By understanding these factors and implementing comprehensive validation strategies, researchers can resolve conflicting results and gain more accurate insights into CTHRC1 biology.

What are the optimal immunohistochemistry protocols for CTHRC1 detection in tissue samples?

For optimal CTHRC1 detection in tissue samples by immunohistochemistry:

Tissue Preparation:

• Use paraformaldehyde-fixed, paraffin-embedded tissues

• Cut sections at standard thickness (4-5 μm)

Antigen Retrieval:

• Heat-mediated antigen retrieval in 10 mmol/L citrate buffer for 15 minutes is essential for detecting CTHRC1

• Allow sections to cool to room temperature before proceeding

Antibody Selection and Dilution:

• For detecting full-length (unprocessed) CTHRC1: Use antipro serum at 1:500 dilution

• For general CTHRC1 detection: Start at 5 μg/mL and optimize as needed

• Include appropriate controls with preimmune serum (1:500 dilution) to assess non-specific staining

Visualization Systems:

• Standard secondary antibody detection systems are suitable

• DAB (3,3'-diaminobenzidine) is commonly used for chromogenic detection

• For fluorescent detection, select secondary antibodies with minimal cross-reactivity

Tissue-Specific Considerations:

• CTHRC1 shows distinct localization patterns in different tissues:

  • Intracellular localization in differentiated smooth muscle cells

  • Reduced expression in dedifferentiated smooth muscle of developing neointima

  • Expression in neurons but not in embryonic myocardium

How do the molecular weight variations of CTHRC1 impact Western blot protocols?

CTHRC1 exhibits complex molecular weight patterns that require specific considerations for Western blot analysis:

Expected Molecular Weights:

• Predicted size: 26-27 kDa (monomeric form)

• Observed sizes: 25-30 kDa (primary band)

• Higher molecular weight forms: dimer (~52 kDa), trimer (~78 kDa)

• Processed fragments: multiple bands below 30 kDa (fragments b, c, d)

• High molecular weight complexes: >130 kDa (in tissue samples under non-reducing conditions)

Protocol Adjustments:

• Sample Preparation:

  • For comprehensive analysis, prepare samples under both reducing and non-reducing conditions

  • For cell lines: NETN lysis buffer has been validated for effective extraction

  • For detecting secreted forms: collect and concentrate conditioned media

• Gel Selection:

  • Use gradient gels (4-20%) to resolve both low and high molecular weight forms

  • For high-resolution separation of fragments: consider using higher percentage gels (12-15%)

• Transfer Conditions:

  • For comprehensive detection of all forms, optimize transfer time and buffer composition

  • For high molecular weight complexes: extend transfer time or use semi-dry transfer systems

• Antibody Selection:

  • Different antibodies may preferentially detect specific forms:

    • Antipro antibodies: detect full-length unprocessed CTHRC1

    • Clone 16D4: recognizes a band in the 50 kDa range under both reduced and non-reduced conditions

    • Some antibodies detect only the larger isoform of CTHRC1

Validation Strategy:

• Include positive controls (recombinant protein, cell lines with known CTHRC1 expression)

• When comparing samples, standardize protein loading (typically 50 μg per lane)

• Consider exposure time optimization (10 seconds has been reported as effective)

What are the best approaches for detecting CTHRC1 protein-protein interactions?

To investigate CTHRC1 protein-protein interactions, several complementary approaches have been validated:

Immunoprecipitation (IP):

• Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• Validated cell lines for IP include UACC-62 and A375 cells

• For analysis of immunoprecipitates, Western blot with alternative anti-CTHRC1 antibodies can confirm specificity

Co-Immunoprecipitation (Co-IP):

• Effective for identifying binding partners of CTHRC1

• Consider using crosslinking reagents to stabilize transient interactions

• Both cell lysates and concentrated conditioned media can be used depending on whether investigating intracellular or secreted complex formation

Analysis of Multimeric Forms:

• Compare non-reducing and reducing conditions to identify covalent and non-covalent interactions

• In non-reduced CHO cell lysates, antibodies recognize bands consistent with monomer, dimer, trimer, and tetramer forms

• In aortic samples, high molecular weight complexes (>130 kDa) can be detected under non-reducing conditions

Plasmin Cleavage Analysis:

• Purified His-tagged CTHRC1 from CHO cells can be used to study proteolytic processing

• Incubation with human plasmin generates specific fragments that can be identified by N-terminal sequencing

• For studying endogenous processing, inhibitors like ε-aminocaproic acid can block plasmin activity

These approaches, used individually or in combination, can provide insights into CTHRC1's interactions with other proteins and its processing mechanisms in different cellular contexts.

How can CTHRC1 antibodies be utilized in cancer biomarker and therapeutic target research?

CTHRC1 has emerged as a potential biomarker and therapeutic target in cancer research, with antibodies playing crucial roles in these investigations:

Biomarker Applications:

• CTHRC1 expression predicts tumor recurrence in prostate cancer, particularly when analyzed alongside PD-1/PD-L1 expression

• Serves as an independent prognostic marker in gastric cancer

• Overexpression detected in multiple cancers including pancreatic, hepatocellular, gastric, colon, and lung cancers

Therapeutic Target Research:

• An anti-CTHRC1 humanized monoclonal antibody has been developed (Patent KR102487687B1)

• For therapeutic antibody development research, understanding CTHRC1's oligomeric forms is crucial - the protein exists as a mixture of monomer (26 kD, 25%), dimer (52 kD, 27%), and trimer (78 kD, 48%)

• Average molecular weight of the CTHRC1 mixture has been calculated as 57.98 kD, with a molar concentration of 100 ng/mL CTHRC1 equivalent to 1.7 nM

Methodological Approaches:

• Antibody-mediated inhibition studies:

  • Comparing effects of anti-CTHRC1 antibodies versus isotype controls (e.g., Human IgG1 isotype control)

  • Evaluating antibody effects on cancer cell migration, invasion, and metastasis

• Functional domains mapping:

  • Using domain-specific antibodies to identify critical regions for CTHRC1's pro-tumorigenic effects

  • Particularly important for CTHRC1's role in pancreatic stellate cells and tumor microenvironment modulation

• Biomarker validation:

  • Standardized immunohistochemistry protocols for tissue microarrays

  • Correlation with clinical outcomes data

What are the current challenges in detecting CTHRC1 in fibrotic disease models?

Detecting CTHRC1 in fibrotic disease models presents unique challenges that researchers should address:

Technical Challenges:

• Variable expression patterns:

  • CTHRC1 shows tissue-specific expression and processing

  • In the brain, CTHRC1 primarily corresponds to fragment a

  • In the lung, fragment b is prevalent

  • During wound healing, CTHRC1 is processed into fragments c and d

• Complex regulation:

  • CTHRC1 is induced by BMP-2 and blocks TGF-β-induced collagen type I and III synthesis

  • Pirfenidone attenuates lung fibrotic fibroblast responses to TGF-β1, potentially involving CTHRC1 pathways

  • NEDD4L-induced β-catenin ubiquitination suppresses interstitial pulmonary fibrosis via inhibiting the CTHRC1/HIF-1α axis

Methodological Solutions:

• Model-specific approaches:

  • For airway remodeling and inflammation in asthma models: CTHRC1 deficiency has protective effects that should be considered when designing experiments

  • For interstitial pulmonary fibrosis: examine both in vitro and in vivo models to capture complex interactions

• Multi-antibody detection strategy:

  • Use antibodies targeting different epitopes to capture the full spectrum of CTHRC1 forms

  • Domain-specific antibodies can help distinguish between processed and unprocessed forms

• Functional correlation:

  • Combine antibody detection with functional assays measuring collagen synthesis

  • CTHRC1 functions as a negative regulator of collagen matrix deposition

  • Consider the role of CTHRC1 in vascular remodeling and tissue repair processes

By addressing these challenges with appropriate experimental designs and antibody selection strategies, researchers can better understand CTHRC1's complex roles in fibrotic diseases.