NOSTRIN Antibody

What is NOSTRIN and why is it significant in research?

NOSTRIN is a 506-amino acid protein of approximately 58 kDa that functions as a key regulator of endothelial Nitric Oxide Synthase (eNOS). It contains an N-terminal cdc15 domain and a C-terminal SH3 domain that are critical for its function . NOSTRIN is significant in research because it contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS, thereby influencing nitric oxide (NO) production and release .

Additionally, NOSTRIN has been identified as having anti-angiogenic, anti-invasive, and anti-inflammatory properties, making it relevant to cancer research . Its expression is particularly abundant in highly vascularized tissues including placenta, kidney, lung, heart, and notably in the colon . Recent studies have shown that NOSTRIN expression is inversely related to survival outcomes in pancreatic ductal adenocarcinoma patients, suggesting its potential as a prognostic marker .

How does NOSTRIN interact with eNOS and affect NO production?

NOSTRIN interacts with eNOS primarily through its SH3 domain. Detailed experiments using deletion constructs have demonstrated that NOSTRIN's C-terminal segment (residues 433-506), which largely represents its SH3 domain, mediates complex formation with eNOS . The binding site on eNOS appears to be located within residues 98-366 of the oxygenase domain, which also contains the caveolin-binding site (residues 350-358) .

When NOSTRIN is overexpressed, it induces a profound redistribution of eNOS from the plasma membrane to vesicle-like structures within the cytoplasm. This relocalization coincides with a significant inhibition of NO release. Specifically, studies in CHO-eNOS cells showed that NOSTRIN overexpression decreased calcium ionophore (A23187)-induced NO production by 62-67% compared to control cells . This mechanism suggests that NOSTRIN regulates eNOS activity not only through direct protein interaction but also by altering its subcellular localization.

What validation methods ensure NOSTRIN antibody specificity?

When using NOSTRIN antibodies, validation of specificity is critical for reliable results. Multiple approaches should be employed:

• Western blot analysis: Using lysates from cells known to express NOSTRIN (e.g., HUVECs, HMVECs) compared with negative controls or NOSTRIN-knockdown samples. A specific antibody should detect a band at approximately 58 kDa .

• Immunoprecipitation validation: Performing reciprocal co-immunoprecipitation experiments where antibodies to NOSTRIN should precipitate NOSTRIN along with interacting proteins like eNOS, and vice versa .

• Immunolocalization studies: Using immunofluorescence to confirm the expected subcellular distribution pattern of NOSTRIN, which should match previously published localization data, showing plasma membrane association in endothelial cells and redistribution upon overexpression .

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide or recombinant NOSTRIN protein, which should abolish specific staining.

The search results indicate that various NOSTRIN antibodies have been generated using different immunogens, including GST-fusion proteins with NOSTRIN 242-506, full-length NOSTRIN 1-506, and synthetic peptides like NOSTRIN 170-186 . Each of these antibodies may have slightly different specificity profiles, making validation particularly important.

How can NOSTRIN antibodies be used to study its role in cancer progression?

NOSTRIN antibodies offer valuable tools for investigating this protein's involvement in cancer progression, particularly in colorectal cancer (CRC) where NOSTRIN is abundantly expressed. Researchers can utilize these antibodies in several sophisticated approaches:

• Tissue microarray analysis: NOSTRIN antibodies can be employed for immunohistochemical staining of human cancer tissue arrays containing samples from various disease stages. Studies have shown significant decreases in NOSTRIN expression with the initiation and progression of advanced colon cancer, suggesting its potential as a prognostic marker .

• Co-immunoprecipitation studies: NOSTRIN antibodies can be used to identify novel protein interactions in cancer cells. For instance, research has demonstrated that NOSTRIN forms an immune complex with Cdk1 in CRC cells and increases the inhibitory Y15 and T14 phosphorylation of Cdk1, thereby halting cytokinesis .

• Correlation with EMT markers: By combining NOSTRIN antibody staining with epithelial-mesenchymal transition (EMT) markers in cancer tissues or cells, researchers can investigate the relationship between NOSTRIN levels and the EMT process, which is crucial for cancer metastasis .

• Cancer stemness studies: NOSTRIN antibodies can help examine the relationship between NOSTRIN expression and cancer stemness markers (CD133, CD44, EpCAM). Research has shown that stable overexpression of NOSTRIN in CRC cell lines decreases expression of these stemness markers and inhibits colonosphere formation .

What is the relationship between NOSTRIN and cell cycle regulation in cancer?

NOSTRIN plays an intriguing role in cell cycle regulation, particularly through its interaction with Cyclin-dependent kinase 1 (Cdk1). Research has revealed that:

• NOSTRIN forms an immune complex with Cdk1 in colorectal cancer cells .

• This interaction results in increased inhibitory phosphorylation of Cdk1 at residues Y15 and T14, which serves to halt cytokinesis .

• The cell cycle regulatory function of NOSTRIN appears to be consistent with its role in inhibiting self-renewal of cancer cells, as demonstrated by decreased BrdU incorporation in cells overexpressing NOSTRIN .

When investigating this relationship, researchers should use NOSTRIN antibodies in combination with phospho-specific Cdk1 antibodies to analyze how NOSTRIN levels correlate with the phosphorylation status of Cdk1. Co-immunoprecipitation experiments with NOSTRIN antibodies followed by western blotting for Cdk1 can confirm this interaction in different cancer cell types or patient samples.

The cell cycle regulatory function of NOSTRIN likely contributes to its reported anti-cancer properties, as aberrant cell cycle progression is a hallmark of cancer progression .

How does NOSTRIN affect stemness and self-renewal in cancer cells?

NOSTRIN has been found to significantly impact cancer cell stemness and self-renewal capabilities. Research using NOSTRIN antibodies has revealed:

• Reduced colonosphere formation: Stable overexpression of NOSTRIN in CRC cell lines curtails their ability to form colonospheres, which are three-dimensional structures formed by stem-like cancer cells .

• Decreased stemness marker expression: NOSTRIN overexpression leads to reduced expression of key cancer stemness markers, including CD133, CD44, and EpCAM .

• Inhibition of self-renewal: BrdU incorporation assays have confirmed NOSTRIN's role in inhibiting self-renewal capabilities of cancer cells .

This suggests that NOSTRIN may function as a tumor suppressor by inhibiting the cancer stem cell phenotype. When investigating this aspect, researchers should use NOSTRIN antibodies in flow cytometry or immunofluorescence studies to correlate NOSTRIN expression with stemness markers at the single-cell level. Additionally, NOSTRIN antibodies can be used to track changes in NOSTRIN expression during differentiation of cancer stem cells or in response to treatments that affect stemness.

What are the optimal conditions for using NOSTRIN antibodies in Western blotting?

Based on published protocols, the following conditions are recommended for optimal Western blotting with NOSTRIN antibodies:

• Sample preparation:

  • Cells should be lysed in stringent buffers containing SDS, as NOSTRIN has been reported to be "hardly soluble in mild detergents such as Triton X-100" .

  • A recommended lysis buffer is RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris⋅HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) .

  • Include protease inhibitors to prevent degradation.

• Gel electrophoresis:

  • Use 10% SDS-PAGE gels for optimal separation of NOSTRIN (58 kDa) .

  • Load equal amounts of protein (20-50 μg) per lane.

• Transfer conditions:

  • Transfer to nitrocellulose membranes at 100V for 1-2 hours or 30V overnight at 4°C.

• Blocking and antibody incubation:

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

  • Primary antibody dilutions: Use α-NOSTRIN antibodies at 1:1000 (for AS574) or 1:5000 (for AS619) dilution .

  • Incubate with primary antibody overnight at 4°C.

  • Secondary antibody: HRP-conjugated anti-rabbit or anti-mouse IgG at 1:5000-1:10000 dilution.

• Detection:

  • Use enhanced chemiluminescence (ECL) for detection.

  • NOSTRIN should appear as a band at approximately 58 kDa.

How can NOSTRIN antibodies be effectively used in immunohistochemistry?

For effective immunohistochemical detection of NOSTRIN in tissue samples, follow these methodological guidelines:

• Tissue preparation:

  • Use paraffin-embedded tissue sections (4 μm thickness) .

  • Perform antigen retrieval to expose epitopes masked during fixation.

• Antigen retrieval:

  • Use citrate buffer (pH 6.0) and heat-induced epitope retrieval.

  • Boil sections in retrieval solution for 15-20 minutes.

• Blocking and antibody incubation:

  • Block with 1% BSA/0.1% Tween 20 in PBS (BPT) .

  • Primary antibody: Incubate with α-NOSTRIN antibodies (e.g., α-NOSTRIN 604) at 1:25 dilution for 1 hour at 37°C .

  • Secondary antibody: Use biotinylated secondary antibody at 1:20 dilution for 30 minutes at room temperature.

• Detection system:

  • Apply FITC- or AMCA-conjugated avidin for visualization .

  • Alternatively, use an HRP-based detection system with DAB as chromogen.

• Controls:

  • Include positive control tissues known to express NOSTRIN (e.g., normal colon tissue).

  • Use corresponding preimmune serum as a negative control.

  • Include no-primary-antibody controls to assess non-specific binding.

• Analysis:

  • Examine with appropriate microscopy (e.g., Zeiss Axioskop II) .

  • Document NOSTRIN expression patterns across different tissue types or disease stages.

What are the best approaches for co-immunoprecipitation experiments with NOSTRIN antibodies?

Co-immunoprecipitation (co-IP) is valuable for studying NOSTRIN's protein interactions. Based on published protocols, follow these guidelines:

• Cell lysis conditions:

  • Use RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris⋅HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) for 1 hour on ice .

  • Remove insoluble material by centrifugation at 13,000 × g for 20 minutes at 4°C.

  • For better solubilization, dilute lysates 1:3 with washing buffer (50 mM Tris⋅HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) .

• Antibody incubation:

  • Incubate diluted lysates with NOSTRIN antibodies (e.g., α-NOSTRIN 532) at a concentration of 10 μl per sample for 1 hour at 4°C under rotation .

  • Include appropriate controls (preimmune sera or non-specific IgG).

• Immunoprecipitation:

  • Precipitate immune complexes with Pansorbin or Protein A/G beads.

  • Centrifuge at 8,000 × g for 2 minutes at 4°C .

  • Wash pellets thoroughly (at least three times) with washing buffer.

• Analysis of immunoprecipitates:

  • Dissolve pellets in sample buffer and separate by SDS-PAGE.

  • For detecting NOSTRIN, immunoblot with α-NOSTRIN 619 at 1:5,000 dilution .

  • For detecting interaction partners (e.g., eNOS), use appropriate antibodies (e.g., α-eNOS m at 1:1,000) .

This approach has successfully demonstrated the interaction between NOSTRIN and eNOS, as well as with Cdk1 in cancer cells .

Why might NOSTRIN antibodies show different staining patterns in different cell types?

NOSTRIN antibodies may show variable staining patterns across different cell types due to several biological and technical factors:

• Differential expression and localization:

  • NOSTRIN's subcellular localization changes depending on cellular context. In confluent human umbilical venous endothelial cells (HUVECs), NOSTRIN colocalizes extensively with eNOS at the plasma membrane, while in CHO-eNOS cells, it appears in punctate cytosolic structures .

  • NOSTRIN's distribution changes upon overexpression, shifting from the plasma membrane to vesicle-like structures .

• Protein interaction partners:

  • NOSTRIN's localization is influenced by its interaction partners, which may vary between cell types.

  • In cells with high eNOS expression, NOSTRIN's localization may be driven by eNOS distribution.

  • NOSTRIN also interacts with N-WASP and dynamin, which could affect its localization pattern .

• Oligomerization state:

  • NOSTRIN can form oligomers (trimers), which may affect epitope accessibility and staining patterns .

  • The oligomerization state might differ between cell types or under different cellular conditions.

• Fixation and permeabilization effects:

  • Different cell types may require adjusted fixation protocols. For instance, published protocols used methanol fixation for HUVECs, HMVECs, and CHO-eNOS cells .

  • The complex between NOSTRIN and eNOS is partially resistant to stringent lysis conditions, suggesting that gentle fixation methods may be required to preserve certain interactions .

When observing different staining patterns, researchers should systematically validate their findings using multiple antibodies targeting different NOSTRIN epitopes and complementary techniques such as fluorescent protein tagging.

What are common pitfalls in NOSTRIN antibody-based research and how can they be addressed?

Several challenges may arise when using NOSTRIN antibodies in research:

• Low solubility issues:

  • NOSTRIN is "hardly soluble in mild detergents such as Triton X-100" .

  • Solution: Use stringent lysis buffers containing SDS (e.g., RIPA buffer) for efficient extraction.

  • For co-IP experiments where harsh detergents might disrupt protein interactions, optimize detergent concentration or use crosslinking approaches.

• Inconsistent detection in Western blots:

  • NOSTRIN may show variable band patterns due to post-translational modifications or degradation.

  • Solution: Include protease inhibitors in all buffers and maintain samples at 4°C throughout processing.

  • Use freshly prepared samples whenever possible and avoid repeated freeze-thaw cycles.

• High background in immunofluorescence:

  • Non-specific binding can obscure true NOSTRIN localization.

  • Solution: Optimize blocking conditions (1% BSA/0.1% Tween 20 in PBS has been effective) .

  • Perform thorough washing steps and include appropriate controls (preimmune sera).

  • Use monoclonal antibodies if polyclonal antibodies show high background.

• Variable expression levels across tissues:

  • NOSTRIN expression varies significantly between tissues, being highest in highly vascularized organs .

  • Solution: Adjust antibody concentrations according to the expected expression level in each tissue type.

  • Include positive control tissues (e.g., placenta, kidney, lung, heart) with known high NOSTRIN expression.

• Difficulties detecting NOSTRIN-protein interactions:

  • Some interactions may be transient or context-dependent.

  • Solution: Consider using in situ proximity ligation assays to detect interactions in intact cells.

  • Use chemical crosslinking before cell lysis to stabilize transient interactions.

  • For studying interactions with the SH3 domain, remember that NOSTRIN forms oligomers which may allow multiple proteins to interact simultaneously via this domain .

Addressing these pitfalls requires careful optimization and validation of protocols for each specific application and cell/tissue type.

How can NOSTRIN expression be used as a potential diagnostic or prognostic marker?

NOSTRIN expression patterns show promising potential as diagnostic and prognostic markers in several cancer types:

• Colorectal cancer progression:

  • Studies using human colon cancer tissue arrays have demonstrated "significant decrease in NOSTRIN expression with initiation and progression of advanced colon cancer disease stages" .

  • This suggests that NOSTRIN antibodies could be used to assess CRC progression and potentially predict outcomes.

• Pancreatic cancer prognosis:

  • NOSTRIN has been identified as part of a 36-gene signature that predicts clinical outcomes in pancreatic ductal adenocarcinoma (PDAC) .

  • Specifically, "NOSTRIN down-regulation was associated with poor outcome" in PDAC patients .

• Correlation with treatment response:

  • NOSTRIN's involvement in gemcitabine-induced apoptotic cell death in pancreatic cancer suggests it might predict treatment response .

  • The combined effect of NOS inhibitors with 5-fluorouracil on CRC cell proliferation indicates that NOSTRIN levels might influence chemotherapy efficacy .

When using NOSTRIN antibodies for clinical research:

• Standardize staining protocols to ensure consistency across patient samples.

• Develop scoring systems based on staining intensity and distribution.

• Correlate NOSTRIN expression with established clinical parameters and patient outcomes.

• Consider combining NOSTRIN assessment with other molecular markers for improved prognostic value.

The potential of NOSTRIN as a biomarker is particularly significant given its "broad biological implications, as enhanced NOSTRIN expression leading to compromised EMT and decreased stemness of cancer cells might inhibit tumorigenesis and colorectal cancer progression" .

What methodologies are most effective for analyzing NOSTRIN expression in patient samples?

For clinical research applications, several complementary methodologies can be employed to analyze NOSTRIN expression in patient samples:

• Immunohistochemistry (IHC):

  • Most widely used method for analyzing protein expression in clinical specimens.

  • Protocol: Use paraffin-embedded tissue sections (4 μm), perform antigen retrieval, and incubate with NOSTRIN antibodies at optimized dilutions (e.g., 1:25 for α-NOSTRIN 604) .

  • Advantage: Preserves tissue architecture, allowing assessment of NOSTRIN localization within the tumor microenvironment.

  • Consider using automated staining platforms for consistency across large sample cohorts.

• Tissue microarray (TMA) analysis:

  • Enables high-throughput analysis of multiple patient samples simultaneously.

  • Has been successfully used to demonstrate decreased NOSTRIN expression with CRC progression .

  • Standardize scoring systems (e.g., H-score or Allred score) to quantify NOSTRIN expression.

• Quantitative PCR:

  • Complements protein-level analysis by measuring NOSTRIN mRNA expression.

  • Can be performed on fresh-frozen tissues or FFPE samples with appropriate RNA extraction methods.

  • Useful for validating IHC findings and providing quantitative expression data.

• Western blotting of clinical specimens:

  • Can provide semi-quantitative assessment of NOSTRIN protein levels.

  • Requires fresh or frozen tissue samples with careful protein extraction.

  • Remember that NOSTRIN requires stringent lysis conditions (e.g., RIPA buffer) for efficient extraction .

• Multiplex immunofluorescence:

  • Allows simultaneous detection of NOSTRIN with other relevant markers (e.g., EMT markers, stemness markers).

  • Provides insights into co-expression patterns within individual cells.

  • Requires careful antibody validation to ensure specificity in multiplex settings.

For comprehensive clinical studies, combining multiple methodologies provides the most robust assessment of NOSTRIN expression and its relationship to disease parameters and patient outcomes.