CSRNP1 Antibody

What is CSRNP1 and why is it significant in research?

CSRNP1 (Cysteine-serine-rich nuclear protein 1), also termed AXUD1 (axin1 up-regulated 1), is a highly conserved nuclear protein that plays critical roles in various biological processes including cell proliferation, cell cycle regulation, and apoptosis. Its significance stems from its involvement in multiple pathological conditions. CSRNP1 has been identified as a key regulator in hepatic ischemia-reperfusion injury (HIRI), where increased expression correlates with tissue damage and hepatocyte apoptosis . Additionally, altered CSRNP1 expression has been linked to hypoxic-ischemic encephalopathy, non-alcoholic fatty liver disease, and serves as a prognostic biomarker in clear cell renal cell carcinoma (ccRCC) . The protein functions downstream of various cytokine signaling pathways and can regulate gene expression through binding to AP-1 consensus-like sequences . This positions CSRNP1 as an important research target for understanding disease mechanisms and developing potential therapeutic strategies.

What types of CSRNP1 antibodies are available for research applications?

Researchers can access several types of CSRNP1 antibodies for different experimental applications:

• Polyclonal antibodies: These are commonly available, such as rabbit polyclonal antibodies that recognize endogenous levels of total CSRNP1 protein . These antibodies are often generated against specific immunogens, such as recombinant human CSRNP1 protein (amino acids 1-200) .

• Application-specific antibodies: Different antibodies may be optimized for specific techniques:

  • Western blot/immunoblotting antibodies (for whole cell lysates or nuclear fractions)

  • Immunohistochemistry (IHC) antibodies

  • Chromatin immunoprecipitation (ChIP) compatible antibodies

The specific validation and reactivity of these antibodies should be carefully considered. For instance, some CSRNP1 antibodies demonstrate reactivity across species (human, mouse, rat), while others may be species-specific . When selecting an antibody, researchers should verify that it has been validated for their intended application and target species.

What are the key experimental considerations when using CSRNP1 antibodies?

When working with CSRNP1 antibodies, researchers should consider several important factors:

• Subcellular localization: CSRNP1 is predominantly nuclear, present in both soluble nuclear fractions and chromatin-bound fractions . Therefore, proper cell fractionation techniques are essential when studying CSRNP1. Standard whole-cell lysates may not provide optimal results compared to nuclear extracts.

• Antibody specificity: Confirm antibody specificity using appropriate controls. Research shows that different CSRNP1 antibodies may have distinct utility depending on the type of sample preparation (e.g., some antibodies work better with nuclear lysates, while others are optimized for whole cell lysates) .

• Expression dynamics: CSRNP1 shows dynamic expression patterns in response to stimuli. For instance, in chondrocytes treated with IL-1 and OSM, CSRNP1 expression peaks at approximately 1.25 hours at the mRNA level and 3 hours at the protein level . These temporal dynamics should inform experimental design and sample collection timepoints.

• Detection methods: Choose detection methods appropriate for nuclear proteins, which may require specialized extraction buffers containing DTT and protease inhibitors .

How should researchers optimize Western blot protocols for CSRNP1 detection?

Optimizing Western blot protocols for CSRNP1 detection requires specific considerations due to its nuclear localization and expression dynamics:

• Sample preparation:

  • For optimal detection, utilize nuclear extraction rather than whole cell lysates

  • Employ nuclear extraction kits (e.g., NE-PER Nuclear and Cytoplasmic Protein Extraction Kit) for efficient isolation

  • Include protease inhibitors (1 mini protease inhibitor cocktail tablet/10 mL buffer) to prevent degradation

  • Add reducing agents such as DTT (1 mM) to maintain protein integrity

• Electrophoresis conditions:

  • Use appropriate percentage gels based on CSRNP1's molecular weight

  • Consider using gradient gels (4-12%) for better resolution

  • Load adequate amounts of nuclear protein (15-30 μg) for clear detection

• Transfer and detection optimization:

  • Employ PVDF membranes rather than nitrocellulose for stronger protein binding

  • Use 5% non-fat milk or BSA in TBS-T for blocking (optimize based on specific antibody recommendations)

  • Incubate with primary antibody (typically 1:1000 dilution) overnight at 4°C for better sensitivity

  • Consider enhanced chemiluminescence (ECL) for detection, as fluorescence-based methods may yield higher background with nuclear proteins

• Controls and validation:

  • Include positive controls (tissues/cells known to express CSRNP1)

  • Consider siRNA knockdown samples as negative controls to confirm antibody specificity

  • Verify band size corresponds to expected molecular weight of CSRNP1

Following stimulation with inflammatory cytokines like IL-1 and OSM, CSRNP1 protein expression peaks at approximately 3 hours and persists up to 24 hours in nuclear fractions , which should guide experimental timepoints.

What are the optimal conditions for immunohistochemical detection of CSRNP1?

For successful immunohistochemical detection of CSRNP1 in tissue samples, researchers should consider the following protocol optimizations:

• Tissue fixation and processing:

  • Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used

  • Optimal fixation time (typically 24-48 hours) is critical to preserve nuclear antigens

  • Consider testing both FFPE and frozen sections to determine optimal preservation of CSRNP1 epitopes

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) is recommended using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Test both buffer systems to determine which provides optimal staining for CSRNP1

  • Typically, 20 minutes at 95-98°C in a pressure cooker yields good results for nuclear antigens

• Antibody dilution and incubation:

  • Test a range of dilutions for the primary antibody (starting with manufacturer recommendations)

  • Extended primary antibody incubation (overnight at 4°C) may improve sensitivity

  • Use appropriate detection systems (e.g., polymer-based detection systems) for enhanced signal

• Controls and validation:

  • Include positive control tissues known to express CSRNP1 (e.g., liver tissue from HIRI models)

  • Use negative controls (primary antibody omission, isotype controls)

  • Consider validating IHC results with other techniques such as in situ hybridization

• Signal amplification:

  • For low-abundance expression, consider using tyramide signal amplification (TSA) to enhance sensitivity

  • Adjust amplification time based on expression levels to avoid background

Since CSRNP1 expression can vary significantly based on physiological conditions (e.g., significantly increased after liver transplantation or in HIRI models) , careful consideration of the experimental context is crucial for interpreting IHC results.

How can researchers effectively use CSRNP1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) with CSRNP1 antibodies requires specific optimization for successful detection of DNA-protein interactions:

• Cross-linking optimization:

  • Standard formaldehyde cross-linking (1% for 5-10 minutes at room temperature) is typically effective

  • Quench with 0.125 M glycine for 5 minutes with agitation

  • For CSRNP1-DNA interactions, avoid over-fixation which may mask epitopes

• Chromatin shearing parameters:

  • Optimize sonication conditions (e.g., 15 cycles of 30 seconds on, 30 seconds off at full power using a Bioruptor or similar device)

  • Aim for DNA fragments of 200-500 bp for optimal resolution

  • Verify shearing efficiency by agarose gel electrophoresis before proceeding

• Immunoprecipitation conditions:

  • Use 2-5 μg of CSRNP1-specific antibody per ChIP reaction

  • Incubate with chromatin overnight at 4°C with rotation

  • Include appropriate controls (IgG control, input control)

  • Consider using protein A/G magnetic beads for cleaner precipitation

• Washing and elution:

  • Perform stringent washing steps to reduce background

  • Elute under appropriate conditions based on antibody specifications

  • Reverse cross-links carefully to preserve DNA integrity

• PCR primers design for target validation:

  • Design primers flanking potential CSRNP1 binding sites

  • For CSRNP1, focus on regions containing AP-1 consensus-like sequences (AGAGTN) with specific attention to promoter regions of target genes like MMP1

Research has shown that CSRNP1 binds preferentially to the AP-1 consensus-like sequences within the proximal promoter region of MMP1 rather than MMP13 , providing a useful positive control target for ChIP experiments.

How should researchers interpret variable CSRNP1 expression patterns across different experimental conditions?

CSRNP1 expression demonstrates significant variability across different experimental conditions and disease states, requiring careful interpretation:

• Temporal expression dynamics:

  • CSRNP1 typically shows biphasic expression patterns with an early transient peak followed by sustained expression

  • In inflammatory stimulation models (e.g., IL-1+OSM in chondrocytes), CSRNP1 mRNA expression peaks at approximately 1.25 hours, while protein levels peak at 3 hours and persist up to 24 hours

  • Unlike other early response factors like cFOS that show transient expression, CSRNP1 expression often persists, suggesting distinct regulatory roles

• Disease-specific expression patterns:

  • In hepatic ischemia-reperfusion injury (HIRI), CSRNP1 expression is significantly upregulated at both mRNA and protein levels

  • In clear cell renal cell carcinoma (ccRCC), CSRNP1 expression correlates with better prognosis and immune infiltration profiles

  • These contrasting roles across different pathologies suggest context-dependent functions

• Correlation with MAPK pathway activation:

  • CSRNP1 expression strongly correlates with MAPK pathway activity scores in liver transplantation samples

  • Researchers should consider measuring related signaling molecules (phosphorylated SAPK, phosphorylated P38 MAPK) alongside CSRNP1 to establish mechanistic connections

• Standardization and normalization approaches:

  • Use appropriate housekeeping genes/proteins for normalization (β-actin for whole cell lysates, histone H3 for nuclear fractions)

  • Consider reporting relative fold changes compared to baseline rather than absolute values

  • When comparing across experimental models, establish clear baseline controls specific to each model

When observing discrepancies in CSRNP1 expression across experiments, researchers should carefully evaluate experimental timepoints, stimulation conditions, and cell/tissue types, as these all significantly impact expression patterns.

What are the common pitfalls in CSRNP1 antibody-based experiments and how can they be resolved?

Researchers frequently encounter several challenges when working with CSRNP1 antibodies:

• False-negative results in Western blots:

  • Problem: Inability to detect CSRNP1 despite expected expression

  • Potential causes: Inadequate nuclear extraction, protein degradation, epitope masking

  • Solutions:

    • Use specialized nuclear extraction protocols rather than standard RIPA buffers

    • Add fresh protease inhibitors to all buffers

    • Verify antibody compatibility with your sample preparation method (some antibodies work better with whole cell lysates, others with nuclear fractions)

• High background in immunohistochemistry:

  • Problem: Non-specific staining obscuring specific CSRNP1 signals

  • Potential causes: Suboptimal blocking, excessive antibody concentration, cross-reactivity

  • Solutions:

    • Increase blocking time and concentration (5% BSA or 10% normal serum)

    • Titrate primary antibody to determine optimal concentration

    • Include absorption controls to confirm specificity

• Inconsistent immunoprecipitation efficiency:

  • Problem: Variable pull-down of CSRNP1 in ChIP or co-IP experiments

  • Potential causes: Inefficient cross-linking, epitope masking, low antibody affinity

  • Solutions:

    • Optimize cross-linking conditions for nuclear proteins

    • Test multiple antibodies targeting different epitopes of CSRNP1

    • Consider tandem purification approaches for challenging interactions

• Conflicting results between experimental models:

  • Problem: CSRNP1 behavior differs between in vitro and in vivo systems

  • Potential causes: Context-dependent regulation, species-specific differences

  • Solutions:

    • Validate findings across multiple experimental systems

    • Consider both acute and chronic models when relevant

    • Verify antibody cross-reactivity when working with different species

For definitive validation of antibody specificity, researchers should consider genetic approaches such as siRNA knockdown of CSRNP1. Studies have shown effective CSRNP1 knockdown using Dharmacon ON-TARGET plus SMARTpool siRNA duplexes (100 nM final concentration) , which can serve as essential negative controls for antibody validation.

How can contradictory findings regarding CSRNP1 function across different studies be reconciled?

The literature reveals apparently contradictory roles for CSRNP1 across different physiological contexts, requiring careful analysis to reconcile these findings:

• Context-dependent effects on cell survival:

  • In hepatic ischemia-reperfusion injury, CSRNP1 inhibition reduces hepatocyte apoptosis through decreased MAPK activation (P38, SAPK)

  • In Drosophila studies, increased CSRNP1 expression disrupts cell cycle progression and promotes apoptosis through JNK activity

  • These contradictions may be reconciled by considering tissue-specific regulatory networks and the timing of CSRNP1 activation

• Divergent prognostic significance:

  • In clear cell renal cell carcinoma, higher CSRNP1 expression is associated with better prognosis and increased immune infiltration

  • In liver injury models, increased CSRNP1 correlates with greater tissue damage

  • This discrepancy highlights the importance of evaluating CSRNP1 in the context of specific disease microenvironments

• Target gene regulation differences:

  • CSRNP1 selectively regulates MMP1 but not MMP13 expression in chondrocytes, despite both genes having AP-1 binding sites

  • This selectivity suggests that CSRNP1 functions within complex transcriptional networks with additional co-factors

To reconcile these contradictions, researchers should:

• Perform comprehensive pathway analyses: Examine CSRNP1 in the context of complete signaling networks rather than in isolation

• Consider temporal dynamics: Assess both immediate and delayed consequences of CSRNP1 modulation

• Account for cell-type specificity: Use cell-type specific approaches (conditional knockouts, cell-specific promoters) when possible

• Validate with multiple methodologies: Combine genetic, pharmacological, and antibody-based approaches

When designing experiments, researchers should clearly define the specific context and anticipated outcome measures, recognizing that CSRNP1 may serve different roles depending on cell type, disease state, and activation dynamics.

How can CSRNP1 antibodies be utilized to investigate its role in signaling pathway crosstalk?

CSRNP1 sits at a junction of multiple signaling networks, particularly involving MAPK pathways. Advanced antibody-based approaches can help elucidate these complex interactions:

• Multiplexed immunofluorescence strategies:

  • Combine CSRNP1 antibodies with antibodies against MAPK pathway components (phospho-P38, phospho-SAPK/JNK, phospho-ERK)

  • Use spectrally distinct fluorophores to visualize co-localization in single cells

  • Quantify correlation coefficients between CSRNP1 and pathway components under various stimulation conditions

  • This approach has revealed that CSRNP1 knockdown reduces phosphorylation of both P38 MAPK and SAPK in hepatocyte models

• Proximity ligation assays (PLA):

  • Apply PLA to detect direct interactions between CSRNP1 and potential binding partners

  • Focus on transcription factors known to associate with AP-1 sites (cJun, cFos, ATF3)

  • Quantify interaction dynamics following stimulation with cytokines or stress conditions

• Sequential ChIP (ChIP-reChIP) methodology:

  • Use sequential immunoprecipitation with CSRNP1 antibodies followed by antibodies against other transcription factors

  • Identify genomic loci where CSRNP1 co-occupies with different partners

  • This approach can help explain the selective binding of CSRNP1 to MMP1 but not MMP13 promoters

• Phospho-specific antibody approaches:

  • Develop or utilize phospho-specific antibodies against CSRNP1 if phosphorylation sites are known

  • Map kinase-specific phosphorylation patterns under different stimulation conditions

  • Correlate phosphorylation status with DNA-binding activity and transcriptional output

Research has demonstrated strong correlation (highest correlation coefficient) between CSRNP1 expression and MAPK activity scores in human liver transplantation samples , suggesting that further investigation of this relationship could provide mechanistic insights into CSRNP1's role in tissue injury.

What experimental approaches can determine the functional consequences of CSRNP1 binding to different genomic regions?

Understanding the functional impact of CSRNP1 genomic binding requires integration of multiple advanced techniques:

• ChIP-Seq combined with RNA-Seq analysis:

  • Perform ChIP-Seq with CSRNP1 antibodies to map genome-wide binding sites

  • Conduct parallel RNA-Seq following CSRNP1 manipulation (overexpression, knockdown)

  • Integrate datasets to identify direct transcriptional targets

  • Focus analysis on genes associated with MAPK signaling and inflammatory responses based on known CSRNP1 functions

• CRISPR-based genomic editing of binding sites:

  • Design CRISPR-Cas9 strategies to mutate specific CSRNP1 binding sites (e.g., the AP-1-like consensus sequence AGAGTN)

  • Compare effects of mutating binding sites in different promoter contexts (e.g., MMP1 vs. MMP13)

  • Measure impact on basal and stimulus-induced gene expression

• DNA-binding affinity measurements:

  • Utilize DNA pull-down assays (DAPA) to assess binding affinity of CSRNP1 to different consensus sequences

  • Research has shown preferential binding of CSRNP1 to AP-1 consensus-like sequences in the MMP1 promoter compared to similar sequences in the MMP13 promoter

  • Quantify binding differences using techniques like surface plasmon resonance or microscale thermophoresis

• Chromatin accessibility analysis:

  • Combine CSRNP1 ChIP-Seq with ATAC-Seq or DNase-Seq

  • Determine whether CSRNP1 binding correlates with changes in chromatin accessibility

  • Assess temporal dynamics of accessibility changes following stimulus-induced CSRNP1 expression

• Transcription factor co-occupancy mapping:

  • Integrate CSRNP1 binding data with publicly available ChIP-Seq datasets for related factors

  • Identify genomic regions where CSRNP1 acts alone versus regions with co-binding of other factors

  • This approach may explain the selective regulation of certain genes (like MMP1) but not others with similar binding motifs

These approaches should be applied across relevant model systems, including hepatocyte models for studying HIRI and chondrocyte models for studying inflammatory gene regulation , to build a comprehensive understanding of context-specific CSRNP1 functions.

How can researchers leverage CSRNP1 antibodies to develop therapeutic strategies for conditions like hepatic ischemia-reperfusion injury?

Translating CSRNP1 research into therapeutic applications requires specialized antibody-based approaches:

• Target validation through in vivo antibody studies:

  • Use cell-permeable antibodies or antibody-mimetic molecules to inhibit CSRNP1 function

  • Administer in preclinical models of hepatic ischemia-reperfusion injury

  • Monitor outcomes including:

    • Serum markers of liver injury (ALT, AST)

    • Histopathological assessment of tissue damage

    • Apoptotic markers (Bax, cleaved caspase-3, Bcl2)

    • MAPK pathway activation (phospho-P38, phospho-SAPK)

• Development of imaging biomarkers:

  • Label CSRNP1 antibodies with imaging agents (fluorophores, radionuclides)

  • Track CSRNP1 expression dynamics in vivo during disease progression

  • Correlate CSRNP1 expression with treatment response and outcome measures

• Combined therapeutic approaches:

  • Test CSRNP1 inhibition in combination with established MAPK inhibitors

  • Research shows that CSRNP1 knockdown reduces MAPK activation, suggesting potential synergistic effects

  • Design therapy timing based on the expression dynamics of CSRNP1 (peaks at early timepoints post-injury)

• Biomarker development for personalized medicine:

  • Develop standardized immunoassays to quantify CSRNP1 levels in patient samples

  • Stratify patients based on CSRNP1 expression patterns

  • In liver transplantation, CSRNP1 is significantly upregulated following transplantation , suggesting potential utility as a biomarker

• Humanized models for translational studies:

  • Use humanized mouse models expressing human CSRNP1

  • Validate antibody targeting strategies in these models

  • Assess efficacy-to-toxicity ratios for various CSRNP1-targeting approaches

These therapeutic development strategies should be informed by the experimental finding that genetic inhibition of CSRNP1 significantly reduces hepatocyte apoptosis in both in vitro hypoxia-reoxygenation models and in vivo HIRI models , suggesting genuine therapeutic potential for CSRNP1 targeting in liver injury.

What is the role of CSRNP1 in immune cell infiltration and inflammatory responses?

Recent research has identified significant correlations between CSRNP1 expression and immune infiltration profiles, opening new research directions:

• T cell subtype regulation:

  • In clear cell renal cell carcinoma (ccRCC), CSRNP1 expression is positively associated with infiltration of type 2 T helper cells in both normal and tumor tissues

  • This association differs from other CSRNP family members (CSRNP2 and CSRNP3), suggesting unique immunomodulatory roles

  • Future research should investigate:

    • Direct effects of CSRNP1 on T cell differentiation and function

    • CSRNP1-dependent chemokine/cytokine production

    • Potential therapeutic targeting to modify immune infiltration patterns

• Inflammation pathway regulation:

  • Functional enrichment analysis has positively associated CSRNP gene family with acute inflammatory response and humoral immune response pathways

  • In liver injury models, CSRNP1 modulates inflammatory cell infiltration and tissue damage

  • Key research questions include:

    • Identification of CSRNP1-regulated inflammatory mediators

    • Temporal relationship between CSRNP1 expression and immune cell recruitment

    • Cell-type specific effects in immune versus parenchymal cells

• Cytokine-induced CSRNP1 expression dynamics:

  • CSRNP1 expression is rapidly induced by cytokines like IL-1 and OSM

  • This suggests a potential feedback loop where initial inflammatory signals induce CSRNP1, which then modulates subsequent inflammatory responses

  • Studies should examine:

    • Comprehensive cytokine response elements in the CSRNP1 promoter

    • Differential induction across tissue-resident immune cell populations

    • Epigenetic regulation of CSRNP1 in chronic inflammatory states

• Comparative immunophenotyping approaches:

  • Multiplex immunohistochemistry using CSRNP1 antibodies alongside immune cell markers

  • Single-cell transcriptomics to correlate CSRNP1 expression with immune cell states

  • Spatial transcriptomics to map CSRNP1-expressing cells relative to immune infiltrates

These research directions could substantially advance our understanding of CSRNP1's role in diseases with inflammatory components, potentially leading to novel immunomodulatory strategies.

How do epigenetic modifications influence CSRNP1 expression and function across different disease contexts?

Emerging evidence suggests epigenetic regulation plays a crucial role in CSRNP1 biology, particularly in disease states:

These epigenetic studies should be conducted across relevant disease models, including liver injury , renal carcinoma , and inflammatory conditions , to identify both common and context-specific regulatory mechanisms.

What are the most promising approaches for generating selective inhibitors or modulators of CSRNP1 function?

Development of specific CSRNP1 modulators represents an exciting frontier in translational research:

• Structure-based drug design approaches:

  • Determine high-resolution structures of CSRNP1, particularly DNA-binding domains

  • Identify druggable pockets and binding sites

  • Design small molecules to disrupt specific protein-protein or protein-DNA interactions

  • Focus on regions that mediate selective DNA binding, such as those distinguishing MMP1 from MMP13 recognition

• Peptide-based inhibitors:

  • Develop peptides mimicking interaction interfaces of CSRNP1 with binding partners

  • Create cell-penetrating peptide conjugates for intracellular delivery

  • Test in cellular models of HIRI to assess antiapoptotic effects

• RNA therapeutic approaches:

  • Optimize siRNA delivery systems building on successful knockdown strategies

  • Develop antisense oligonucleotides targeting CSRNP1 mRNA

  • Explore CRISPR-based transcriptional repression of CSRNP1

  • Assess liver-directed delivery systems for hepatic applications

• Targeted protein degradation:

  • Design CSRNP1-directed PROTACs (proteolysis targeting chimeras)

  • Create molecular glues to induce selective CSRNP1 degradation

  • Assess the impact of rapid CSRNP1 degradation versus transcriptional inhibition

• Conformation-specific antibodies:

  • Develop antibodies that recognize specific functional states of CSRNP1

  • Target epitopes that distinguish activated from inactive CSRNP1

  • Focus on regions involved in MAPK-dependent activation

Each of these approaches should be evaluated based on:

• Selectivity for CSRNP1 over other CSRNP family members

• Effects on physiological versus pathological CSRNP1 function

• Tissue-specific delivery and activity

• Pharmacokinetic and safety profiles in relevant preclinical models

Research has demonstrated that genetic inhibition of CSRNP1 ameliorates hepatic ischemia-reperfusion injury in an MAPK-dependent manner , providing strong rationale for therapeutic development in this direction.