NISCH Antibody

What is Nischarin and what epitopes do NISCH antibodies typically recognize?

Nischarin (NISCH) is a ~166.6 kDa protein that may also be known by alternative designations including I-1, IR1, IRAS, hIRAS, I-1 receptor candidate protein, and I1R candidate protein . The protein has four reported isoforms, with isoform 1 coding for the full-length protein being dominant in both healthy and tumor tissues . Most commercially available NISCH antibodies target regions closer to the N-terminus, which allows them to detect all isoforms . These antibodies are critical tools for examining nischarin expression and localization across diverse experimental contexts.

What are the validated applications for NISCH antibodies in research settings?

NISCH antibodies have been validated for multiple experimental applications. Primary validated applications include Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . When selecting a NISCH antibody, researchers should verify the specific applications for which each antibody has been validated. For example, the mouse monoclonal IgG1 kappa light chain antibody (F-3) has been validated for detecting Nischarin protein from mouse, rat, and human origins across all these applications .

How does Nischarin expression differ between normal and tumor tissues?

Nischarin expression is significantly decreased in most tumor types compared to their healthy tissue counterparts. Proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) showed significantly lower NISCH protein levels in nine out of ten examined tumor types, with pancreatic ductal adenocarcinoma being the only exception where the decrease was present but not statistically significant . This downregulation occurs most commonly due to deletions of the nischarin gene and promoter methylation . Cancer-specific methylation of the NISCH gene has been documented in breast, ovarian, lung, head and neck, and gastric cancers, while NISCH loss of heterozygosity has been reported in breast and ovarian cancers .

What are the optimal methods for studying Nischarin's subcellular localization in different tissue contexts?

For studying these localization patterns, researchers should consider:

• Using immunohistochemistry with validated antibodies such as HPA023189, which recognizes all four NISCH protein isoforms

• Implementing cell fractionation techniques to isolate nuclear, cytoplasmic, and membrane protein fractions before Western blot analysis

• Employing confocal microscopy with co-localization markers for different cellular compartments

• Including appropriate positive and negative controls for each cellular compartment

The subcellular distribution pattern varies significantly by cancer type - for instance, breast cancer and endometrial cancer samples exhibit only cytoplasmic and membranous staining, while colon adenocarcinoma and hepatocellular carcinoma show moderate nuclear, cytoplasmic, and membranous staining .

How should researchers address the context-dependent prognostic value of Nischarin in different cancer types?

The prognostic value of Nischarin varies dramatically across cancer types and even within patient subgroups. For instance, high NISCH expression was associated with better prognosis in lung adenocarcinoma and pancreatic cancer, but correlated with worse outcomes in colon adenocarcinoma and prostate adenocarcinoma . Additionally, in melanoma, NISCH was a favorable prognostic marker only in female patients but not in males .

To properly investigate these context-dependent effects, researchers should:

• Stratify patient cohorts by sex, cancer subtype, stage, and grade

• Perform multivariate analysis to account for confounding factors

• Consider both mRNA and protein expression levels, as these can yield different prognostic associations

• Analyze pathway enrichment to understand the molecular mechanisms underlying differential prognostic associations

• Validate findings across independent cohorts using consistent methodologies and antibodies

What experimental approaches can effectively evaluate the functional significance of Nischarin's nuclear localization?

The nuclear localization of Nischarin in certain tumor types suggests potentially distinct functions that remain poorly understood. To investigate this phenomenon, researchers should consider:

• Cellular fractionation followed by Western blotting to quantify nuclear versus cytoplasmic Nischarin content

• Immunofluorescence microscopy with nuclear counterstains (DAPI/Hoechst) to visualize and quantify nuclear localization patterns

• Creating deletion or mutation constructs of Nischarin's putative nuclear localization signals for transfection experiments

• Chromatin immunoprecipitation (ChIP) assays to identify potential DNA-binding activities or chromatin associations

• Co-immunoprecipitation experiments to identify nuclear-specific protein-protein interactions

• RNA-seq after nuclear Nischarin modulation to identify transcriptional changes

These approaches can help determine whether nuclear Nischarin has functions distinct from its cytoplasmic and membranous roles .

What controls are essential when utilizing NISCH antibodies for cancer research applications?

When designing experiments using NISCH antibodies for cancer research, the following controls are essential:

• Positive controls: Include cell lines or tissues known to express Nischarin (verify from Human Protein Atlas or previously published literature)

• Negative controls: Use NISCH knockout or knockdown cells, or tissues known to have low/no Nischarin expression

• Isotype controls: Include the appropriate isotype-matched control antibody to identify non-specific binding

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate specificity

• Multiple antibody validation: When possible, confirm findings using antibodies from different sources or that recognize different epitopes

• Cross-tissue validation: Include multiple tissue types to account for tissue-specific expression patterns, especially when comparing normal versus tumor samples

These controls help ensure reliable and reproducible results, particularly important given the context-dependent nature of Nischarin expression and localization patterns .

How can researchers optimally design experiments to investigate sex-dependent differences in Nischarin function?

Given the observed sex-dependent prognostic significance of Nischarin in certain cancers like melanoma, researchers should implement the following experimental design considerations:

• Patient cohort selection: Ensure balanced representation of male and female patients and analyze data separately by sex

• Cell line selection: Include cell lines derived from both male and female patients for in vitro studies

• Animal models: Use both male and female animals in preclinical studies, analyzing results separately

• Hormonal considerations: Assess the potential influence of sex hormones by including hormone treatment/blockade experiments

• Gene expression analysis: Perform sex-stratified gene set enrichment analyses to identify sex-specific signaling pathways associated with Nischarin

• Protein interaction studies: Investigate whether Nischarin has sex-specific protein interaction partners

This approach can help elucidate the molecular mechanisms underlying the observed sex differences in Nischarin's prognostic value .

What methodological approaches are recommended for studying Nischarin agonists as potential anti-cancer therapeutics?

Based on findings that Nischarin agonists like rilmenidine can reduce cancer cell viability, researchers investigating Nischarin-targeting therapeutics should consider:

• Cell viability assays: Use multiple assays beyond MTT (such as Annexin-PI for apoptosis detection) to distinguish between cytotoxic, cytostatic, and metabolic effects

• Dose-response and time-course experiments: Test different concentrations over multiple time points to establish optimal treatment parameters

• Cell line panel selection: Include cell lines representing cancers where Nischarin is a positive versus negative prognostic marker to elucidate context-dependent effects

• Mechanism of action studies: Investigate whether agonist effects are mediated through canonical Nischarin pathways by including knockdown/knockout controls

• Combination studies: Test Nischarin agonists in combination with standard-of-care therapies for potential synergistic effects

• In vivo validation: Progress to animal models with careful attention to sex-dependent effects

For example, when studying rilmenidine, researchers observed that it dose-dependently decreased viability in all tested cancer cell lines, with A-375 melanoma cells showing the highest sensitivity and HT-29 colon cancer cells showing the lowest. Follow-up Annexin-PI apoptosis assays confirmed that rilmenidine induced time- and dose-dependent apoptosis in melanoma cells .

How should researchers reconcile discrepancies between NISCH mRNA and protein expression data?

Researchers often encounter discrepancies between mRNA and protein expression data for Nischarin. To address these discrepancies:

• Methodological validation: Ensure both RNA and protein detection methods are properly validated with appropriate controls

• Isoform consideration: Check whether mRNA and protein detection methods target the same isoforms

• Post-transcriptional regulation: Investigate microRNA-mediated regulation or RNA stability factors

• Post-translational modifications: Examine potential protein degradation, stability issues, or modifications affecting antibody recognition

• Subcellular localization: Consider whether protein localization affects detection (e.g., nuclear translocation may affect whole-cell protein quantification)

• Temporal dynamics: Account for potential time lags between transcription and translation

For example, in ovarian cancer, one study reported that higher NISCH mRNA expression was an unfavorable prognostic marker, while another study found that increased NISCH protein levels were associated with better prognosis . Such discrepancies highlight the importance of examining both mRNA and protein levels in multiple independent cohorts.

What analytical approaches can help understand the apparent contradiction of Nischarin as both tumor suppressor and negative prognostic marker?

The seemingly contradictory roles of Nischarin present a significant interpretative challenge. To address this complexity:

• Context-specific analysis: Analyze data within specific cancer types, subtypes, stages, and patient demographics

• Pathway analysis: Perform gene set enrichment analysis to identify context-specific associated pathways (e.g., in tumors where high Nischarin expression is a negative prognostic marker, stemness-related pathways are often enriched)

• Isoform-specific studies: Investigate whether different Nischarin isoforms have distinct functions

• Localization-function correlation: Determine whether nuclear versus cytoplasmic/membranous localization correlates with different functional outcomes

• Interactome analysis: Identify context-specific protein interaction partners that might modify Nischarin function

This multi-faceted approach can help resolve the apparent paradox of Nischarin's dual roles in cancer biology .

What considerations are important when interpreting immunohistochemical data for Nischarin across different tumor types?

When interpreting immunohistochemical data for Nischarin across tumor types, researchers should consider:

• Staining pattern heterogeneity: Account for variations in staining intensity and subcellular localization patterns within and between tumor types

• Quantification methods: Standardize scoring systems for intensity, percentage of positive cells, and subcellular localization

• Batch effects: Control for technical variations between staining batches using proper controls

• Antibody clone specificity: Verify that the antibody used (e.g., HPA023189) recognizes all relevant Nischarin isoforms

• Tumor microenvironment: Consider whether stromal or immune cell staining may confound tumor cell assessment

• Clinical correlation: Correlate staining patterns with comprehensive clinical data, including treatment history and outcome

For example, breast cancer samples exhibit only cytoplasmic and membranous staining, while colon adenocarcinoma and hepatocellular carcinoma show moderate nuclear, cytoplasmic, and membranous staining, with the percentage of samples showing nuclear localization varying from 10% in glioma to 50% in several other cancer types .

What are the most promising approaches for investigating the mechanistic basis of Nischarin's sex-dependent effects in cancer?

To investigate sex-dependent effects of Nischarin, researchers should consider:

• Hormonal regulation studies: Determine whether sex hormones regulate Nischarin expression, localization, or function

• X-chromosome inactivation analysis: Investigate potential sex-specific epigenetic regulation since the NISCH gene is located on chromosome 3p21.1

• Sex-specific transcriptome profiling: Perform RNA-seq in male versus female cancer cells with Nischarin modulation

• Protein-protein interaction mapping: Identify sex-specific interacting partners

• In vivo models: Develop sex-specific xenograft or genetic models with Nischarin modulation

• Clinical trial stratification: Design future clinical studies of Nischarin agonists with sex-stratified analysis plans

These approaches could help elucidate why Nischarin has positive prognostic value in female melanoma patients but negative value in males .

How might researchers develop more specific NISCH antibodies to distinguish between different isoforms and their functions?

Currently available NISCH antibodies typically bind to the N-terminal region and detect all isoforms. To develop isoform-specific antibodies:

• Epitope mapping: Identify unique peptide sequences specific to each isoform

• Custom antibody development: Generate antibodies against isoform-specific epitopes

• Validation strategy: Design comprehensive validation panels including overexpression and knockout controls for each isoform

• Application-specific testing: Validate new antibodies for specific applications (WB, IP, IF, IHC)

• Functional correlation: Correlate isoform-specific detection with functional outcomes in cellular models

Such isoform-specific antibodies would help determine whether the nuclear localization observed in some tumor types is isoform-specific and whether different isoforms have distinct functions in cancer progression .

What experimental paradigms could best evaluate the therapeutic potential of Nischarin agonists across different cancer contexts?

Given that Nischarin expression is negatively associated with pathways controlling cancer growth and progression, FDA-approved Nischarin agonists like rilmenidine represent interesting drug repurposing candidates. To evaluate their therapeutic potential:

• Cancer type prioritization: Focus initial studies on cancers where high Nischarin expression is a positive prognostic marker

• Precision medicine approach: Develop biomarkers to identify patients likely to respond (e.g., Nischarin expression level, localization pattern)

• Combination therapy screening: Test Nischarin agonists with standard chemotherapies and targeted agents

• Mechanism of action studies: Determine whether anti-cancer effects occur through canonical Nischarin pathways or novel mechanisms

• Resistance mechanism investigation: Identify potential resistance pathways to inform combination strategies

• Patient-derived xenograft models: Evaluate efficacy in models that better recapitulate tumor heterogeneity

Early studies have demonstrated that the Nischarin agonist rilmenidine dose-dependently decreased viability in multiple cancer cell lines and induced apoptosis in melanoma cells, suggesting potential therapeutic applications worthy of further investigation .