NRIP2 Antibody

What is NRIP2 and what cellular functions has it been implicated in?

NRIP2 (Nuclear Receptor Interacting Protein 2) is a protein that interacts with nuclear receptors and plays critical roles in gene regulation. Research has established NRIP2 as a key modulator of several important cellular pathways:

• Wnt Signaling Regulation: NRIP2 interacts with the Wnt pathway, particularly by modulating β-catenin stability and activation .

• Nuclear Receptor Interaction: NRIP2 binds to and regulates the activity of nuclear receptors such as retinoic acid-related orphan receptor β (RORβ) .

• Transcriptional Regulation: NRIP2 prevents RORβ from binding to downstream promoter regions of inhibitory factors like HBP1, thereby attenuating HBP1-dependent inhibition of TCF4-mediated transcription .

• Protein Stabilization: NRIP2 interacts with β-catenin, specifically at its N-terminal domain, preventing its degradation through the ubiquitin proteasomal pathway .

Recent studies have shown upregulation of NRIP2 in various pathological conditions, including colorectal cancer initiating cells and podocytes in focal segmental glomerulosclerosis (FSGS) patients , suggesting its involvement in disease progression.

What types of NRIP2 antibodies are commonly used and which applications are they validated for?

Several types of NRIP2 antibodies are available for research, with varying applications based on their validation studies:

Types of NRIP2 antibodies:

• Polyclonal antibodies: Most common, such as rabbit polyclonal anti-NRIP2 antibodies

• Monoclonal antibodies: Used for more specific detection with reduced background

Validated applications include:

When selecting an NRIP2 antibody, researchers should consider the specific epitope recognition and cross-reactivity profile. The rabbit anti-NRIP2 antibody (such as Affinity, AF0597) has been successfully used at 1:100 dilution for IF/IHC applications and 1:500 for Western blotting in recent studies .

How should NRIP2 antibodies be validated before experimental use?

Proper validation of NRIP2 antibodies is crucial for ensuring experimental reproducibility and reliability. Recommended validation procedures include:

• Specificity Testing:

  • Knockout/knockdown controls: Compare antibody signal in NRIP2 knockout or knockdown cells versus wild-type cells

  • Overexpression systems: Test in cells with and without NRIP2 overexpression

  • Peptide blocking: Pre-incubate antibody with the immunizing peptide to confirm specific binding

• Application-Specific Validation:

  • Western blot: Confirm single band of expected molecular weight

  • IHC/IF: Demonstrate proper subcellular localization and absence of signal in negative controls

  • IP: Verify recovery of NRIP2 protein and relevant binding partners (e.g., β-catenin, RORβ)

• Cross-reactivity Assessment:

  • Test on tissues/cells from different species if planning cross-species studies

  • Validate antibody performance in the specific experimental conditions (fixation methods, buffers, etc.)

• Lot-to-Lot Consistency:

  • When receiving a new lot, compare performance to previously validated lot

Researchers studying NRIP2 in kidney disease models have successfully validated antibodies by confirming loss of signal in NRIP2 knockout mice and demonstrating colocalization with established markers like WT1 in podocytes .

What are the molecular mechanisms by which NRIP2 regulates the Wnt/β-catenin signaling pathway?

NRIP2 has been established as a critical regulator of the Wnt/β-catenin pathway through multiple molecular mechanisms:

• Direct Binding to β-catenin:

  • NRIP2 physically interacts with β-catenin's N-terminal domain as demonstrated by co-immunoprecipitation studies

  • This interaction occurs in both physiological and pathological conditions

• Prevention of β-catenin Degradation:

  • NRIP2 prevents β-catenin ubiquitination and subsequent proteasomal degradation

  • NRIP2 knockdown significantly enhances β-catenin ubiquitination, while NRIP2 overexpression or MG132 treatment blocks this process

  • Treatment with the proteasome inhibitor MG132 reverses the effect of NRIP2 knockdown, confirming the proteasome-dependent mechanism

• Indirect Regulation Through RORβ:

  • NRIP2 binds to retinoic acid-related orphan receptor β (RORβ)

  • RORβ normally acts as a transcriptional enhancer of HBP1, which inhibits TCF4-mediated transcription

  • NRIP2 prevents RORβ from binding to the HBP1 promoter regions, reducing HBP1 transcription

  • This leads to attenuated HBP1-dependent inhibition of the Wnt pathway

• Activation of Downstream Target Genes:

  • NRIP2 overexpression leads to increased expression of Wnt target genes including α-SMA, desmin, and Col1a1

  • Silencing β-catenin attenuates these NRIP2-induced effects

These mechanisms suggest that NRIP2 acts as both a direct stabilizer of β-catenin and an indirect regulator of the Wnt pathway through modulation of transcriptional repressors like HBP1.

What experimental models are most suitable for studying NRIP2 function in kidney diseases?

Based on recent research, several experimental models have proven effective for studying NRIP2 function in kidney diseases:

• In Vitro Models:

  • Human podocyte cell lines: Immortalized human podocytes have been successfully used to study NRIP2 function through knockdown and overexpression experiments

  • Adriamycin (ADR) treatment: ADR-treated podocytes serve as an effective model of podocyte injury with demonstrated β-catenin activation

  • MG132 proteasome inhibition: Useful for investigating NRIP2's role in preventing β-catenin degradation

• In Vivo Models:

  • NRIP2 knockout mice: Global allelic knockout of NRIP2 using CRISPR/Cas9 on C57BL/6N background

  • Adriamycin nephropathy model: ADR injection (20 mg/kg body weight via tail vein) in wild-type and NRIP2 KO mice induces podocyte injury and proteinuria

  • Assessment methods: Urine albumin-to-creatinine ratio, histological examination by light microscopy and transmission electron microscopy

• Human Tissue Samples:

  • Kidney biopsy samples from FSGS patients: Valuable for examining NRIP2 expression and colocalization with WT1 and β-catenin

  • Publicly available datasets: Nephroseq database and GEO datasets (e.g., GSE129973) provide expression data across different kidney diseases

Model Selection Considerations:

The use of multiple complementary models is recommended for comprehensive understanding of NRIP2 function in kidney diseases.

What methodological approaches are effective for NRIP2 protein-protein interaction studies?

Investigating NRIP2's interactions with other proteins requires specific methodological approaches. Based on successful studies, the following methods are recommended:

• Co-Immunoprecipitation (Co-IP):

  • Lysate preparation: Use Minute™ Total Protein Extraction Kit (Invent, SN-002) with protease inhibitors for cell lysis

  • Antibody incubation: Incubate lysates with anti-β-catenin or anti-NRIP2 antibody overnight at 4°C on a rotator

  • Protein capture: Add protein A/G Magnetic Beads (15 μl) for 3 hours at 4°C

  • Washing and elution: Wash thoroughly with cold PBS, resuspend in 2× SDS buffer, and boil for 10 minutes

  • Analysis: Subject supernatants to SDS-PAGE and immunoblot for interaction partners

• Ubiquitination Assays:

  • MG132 treatment: Treat cells with 5 μM MG132 for 1 hour before harvesting to prevent proteasomal degradation

  • HA-tagged ubiquitin: Transfect cells with pRK5-HA-ubiquitin plasmid to detect ubiquitination

  • Immunoprecipitation: Pull down β-catenin and probe for ubiquitin to assess ubiquitination levels

  • Controls: Include both NRIP2 knockdown and overexpression conditions

• Domain Mapping:

  • Deletion mutants: Create β-catenin deletion mutants to identify interaction domains with NRIP2

  • Co-transfection: Transfect NRIP2 with different β-catenin deletion constructs

  • Affinity assessment: Compare binding affinities to identify critical interaction domains

• Functional Validation:

  • Knockdown/overexpression validation: Confirm effects using siRNA knockdown (#1: GUGUAGTGTGATUUAAAGA; #2: GAUAGAGATTUUAGUTCTT for human NRIP2)

  • Rescue experiments: Perform rescue experiments by re-introducing NRIP2 after knockdown

  • Colocalization studies: Use immunofluorescence to confirm protein interactions in situ

These methodologies have successfully demonstrated that the N-terminal domain of β-catenin mediates interaction with NRIP2, and that this interaction prevents β-catenin degradation through the ubiquitin proteasomal pathway .

How does NRIP2 expression vary across different pathological conditions and what techniques best capture these differences?

NRIP2 shows differential expression patterns across various pathological conditions, which can be detected through several complementary techniques:

Expression in Pathological Conditions:

• Cancer:

  • Significantly upregulated in colorectal cancer initiating cells (CCICs)

  • Functions as a novel interactor with the Wnt pathway in cancer contexts

• Kidney Diseases:

  • Increased expression in several nephrotic syndromes according to Nephroseq database analysis :

    • Minimal change disease (MCD)

    • Focal segmental glomerulosclerosis (FSGS)

    • Membranous nephropathy (MN)

  • Not significantly altered in IgA nephropathy (IgAN)

  • Predominantly expressed in podocytes in FSGS patients

Optimal Detection Methods:

For comprehensive assessment of NRIP2 in disease contexts, researchers should consider using multiple techniques. In kidney disease studies, the combination of database analysis, western blotting for protein expression, and immunohistochemistry/immunofluorescence for localization has proven particularly effective .

What are the current methodological challenges in NRIP2 functional studies and how can they be addressed?

Researchers face several methodological challenges when investigating NRIP2 function, with potential solutions based on recent successful studies:

• Specificity of Knockdown/Knockout Effects:
  Challenge: Global NRIP2 knockout affects multiple cell types, making it difficult to isolate cell-specific effects.
  Solution:

  • Use cell-specific Cre-loxP knockout systems

  • Complement global knockout studies with cell-specific knockdown experiments

  • As noted in kidney studies: "We could not preclude the effects of NRIP2 loss in cells other than podocytes"

• Understanding the Full Protein Interaction Network:
  Challenge: Identifying all relevant protein interactions of NRIP2.
  Solution:

  • Employ proteomics approaches such as proximity labeling (BioID, APEX)

  • Use systems biology approaches to map the complete interactome

  • Address knowledge gaps noted in recent research: "We did not explore which E3 ubiquitin ligase competes with NRIP2 to recognize β-catenin in podocytes"

• Distinguishing Direct vs. Indirect Effects:
  Challenge: Determining whether NRIP2 effects are direct or mediated through other factors.
  Solution:

  • Use in vitro binding assays with purified proteins

  • Perform chromatin immunoprecipitation studies for transcriptional effects

  • Investigate whether "NRIP2 cooperates with β-catenin to drive target gene expression"

• Translating Between Models and Human Disease:
  Challenge: Ensuring relevance of model findings to human pathology.
  Solution:

  • Validate findings in human samples and patient-derived cells

  • Use organoid cultures and patient-derived xenografts

  • Compare model expression patterns with human disease databases

• Technical Aspects of Protein Detection:
  Challenge: Consistent detection of NRIP2 across samples and studies.
  Solution:

  • Standardize antibody validation procedures

  • Use multiple antibodies targeting different epitopes

  • Include proper positive and negative controls (NRIP2 overexpression and knockout)

Experimental Design Recommendations:

For studies investigating NRIP2's role in the ubiquitin-proteasomal degradation pathway:

• Include both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches

• Use proteasome inhibitors (e.g., MG132 at 5 μM for 1 hour) as controls

• Perform ubiquitination assays with HA-tagged ubiquitin constructs

• Create domain-specific mutants to map functional regions

By addressing these methodological challenges, researchers can gain more comprehensive insights into NRIP2's diverse biological functions and potential therapeutic applications.

How might NRIP2 be targeted for therapeutic purposes in kidney diseases and cancer?

Based on recent findings about NRIP2's role in disease pathways, several therapeutic strategies could be explored:

• Small Molecule Inhibitors:

  • Target the NRIP2-β-catenin interaction interface, particularly focusing on the N-terminal domain of β-catenin that mediates binding to NRIP2

  • Disrupt NRIP2-RORβ binding to restore normal RORβ-mediated transcription of HBP1

  • Design compounds that modulate NRIP2's protein stabilization function without affecting its other biological roles

• Gene Therapy Approaches:

  • Use siRNA or antisense oligonucleotides to reduce NRIP2 expression in affected tissues

  • The siRNA sequences (#1: GUGUAGTGTGATUUAAAGA; #2: GAUAGAGATTUUAGUTCTT) have shown efficacy in cell culture models

  • Targeted delivery systems would be needed for tissue-specific effects

• Biologics Development:

  • Develop neutralizing antibodies against NRIP2

  • Design decoy peptides that mimic NRIP2 binding domains to competitively inhibit pathological interactions

• Combination Therapies:

  • Target NRIP2 together with other Wnt pathway components

  • Combine with proteasome inhibitors to modulate β-catenin stability through multiple mechanisms

• Biomarker Potential:

  • Use NRIP2 expression levels as biomarkers for disease progression

  • Especially relevant in kidney diseases where NRIP2 is upregulated in glomeruli from MCD, FSGS, and MN patients

Therapeutic Potential Based on Disease Context:

Evidence from NRIP2 knockout studies in ADR nephropathy models demonstrates the therapeutic potential, as genetic knockout ameliorated podocyte injury and proteinuria by inhibiting β-catenin activation .

What methodological considerations should be addressed when using NRIP2 antibodies in diagnostic applications?

While NRIP2 antibodies show promise for diagnostic applications, several methodological considerations must be addressed:

• Antibody Selection for Diagnostics:

  • Specificity validation: Complete validation in relevant tissues, including comparison of staining patterns between normal and diseased specimens

  • Reproducibility testing: Assess inter-laboratory and inter-observer variability

  • Epitope considerations: Select antibodies recognizing epitopes that remain accessible in processed clinical samples

• Tissue Processing and Staining Protocols:

  • Fixation optimization: Determine optimal fixation methods that preserve NRIP2 antigenicity

  • Antigen retrieval: Standardize antigen retrieval methods (pH, temperature, duration)

  • Signal amplification: For low-abundance detection, consider tyramide signal amplification or other enhancement methods

  • Blocking protocols: Optimize to reduce background while maintaining specific signal

• Quantitative Assessment Methods:

  • Scoring systems: Develop and validate standardized scoring systems for NRIP2 expression

  • Digital pathology: Employ digital image analysis for objective quantification

  • Multi-marker panels: Integrate NRIP2 with other markers for improved diagnostic accuracy

• Clinical Interpretation Guidelines:

  • Expression thresholds: Establish clinically relevant expression thresholds

  • Pattern recognition: Document subcellular localization patterns that correlate with disease states

  • Control samples: Include appropriate positive and negative controls for each diagnostic run

• Technical Validation for Clinical Use:

  • Sample size considerations: Validate on sufficiently large cohorts to account for biological variability

  • Automation compatibility: Ensure compatibility with automated staining platforms

  • Stability testing: Determine antibody shelf-life and stability under clinical laboratory conditions

For kidney disease applications specifically, research has shown that NRIP2 antibody staining (Affinity, AF0597; IF/IHC: 1:100) can effectively detect elevated NRIP2 expression in podocytes from FSGS patients, with colocalization with WT1 confirming podocyte-specific expression . This suggests potential diagnostic utility, but standardization of these methods would be necessary for clinical implementation.

What are the most promising future research directions for understanding NRIP2 function beyond current knowledge?

Current research has established NRIP2's roles in Wnt signaling, β-catenin stabilization, and podocyte injury, but several promising research directions could significantly expand our understanding:

• Comprehensive Interactome Mapping:

  • Apply proximity labeling technologies (BioID, APEX) to identify the complete NRIP2 protein interaction network

  • Determine tissue-specific and condition-specific interactors

  • Address unresolved questions: "We did not explore which E3 ubiquitin ligase competes with NRIP2 to recognize β-catenin in podocytes"

• Structure-Function Studies:

  • Determine the three-dimensional structure of NRIP2 alone and in complex with β-catenin and RORβ

  • Map functional domains beyond the known interaction regions

  • Investigate conformational changes upon binding to different partners

• Expanded Disease Relevance:

  • Explore NRIP2's role in other Wnt-dependent pathologies beyond colorectal cancer and kidney disease

  • Investigate potential functions in developmental processes

  • Examine involvement in additional signaling pathways beyond Wnt/β-catenin

• Transcriptional Regulatory Mechanisms:

  • Conduct ChIP-seq studies to identify genes directly regulated by NRIP2-containing complexes

  • Investigate whether "NRIP2 cooperates with β-catenin to drive target gene expression"

  • Determine if NRIP2 has DNA-binding capacity or acts exclusively through protein-protein interactions

• Advanced In Vivo Models:

  • Develop tissue-specific and inducible NRIP2 knockout models

  • Create humanized models that recapitulate human NRIP2 expression patterns

  • Investigate NRIP2 function in disease models beyond ADR nephropathy

• Therapeutic Targeting Approaches:

  • Screen for small molecules that specifically disrupt the NRIP2-β-catenin interaction

  • Develop peptide inhibitors that target specific functional domains

  • Explore PROTAC (Proteolysis Targeting Chimera) approaches to induce NRIP2 degradation

• Regulatory Mechanisms Controlling NRIP2:

  • Identify transcription factors that regulate NRIP2 expression

  • Investigate post-translational modifications that alter NRIP2 function

  • Explore potential regulatory non-coding RNAs that modulate NRIP2 expression

These research directions would address significant knowledge gaps and potentially unlock new therapeutic strategies for diseases where NRIP2 dysregulation plays a role.