CAPRIN2 Antibody

What is CAPRIN2 and what are its primary functional roles in cellular processes?

CAPRIN2 is an RNA-binding protein that plays multiple critical roles in cellular function. It contains RNA-binding coiled-coil and RGG box domains that enable its RNA-binding activity, primarily functioning in mRNA regulation and stress granule formation . CAPRIN2 regulates translation through interaction with the eukaryotic initiation factor 3 (eIF3) complex, particularly suppressing translation of longer mRNAs associated with cell proliferation while allowing shorter mRNAs (like crystallins in lens development) to escape this inhibition .

Additionally, CAPRIN2 enhances canonical Wnt signaling by promoting phosphorylation of the Wnt coreceptor LRP6 . In the hypothalamus, CAPRIN2 binds directly to arginine vasopressin (AVP) mRNA, influencing osmotic regulation and fluid homeostasis . CAPRIN2 is also essential for proper eye lens development, where it regulates differentiation of lens fiber cells .

The protein is highly expressed in brain tissue but demonstrates context-dependent functions across different cell types, making it a multifunctional regulator of both RNA metabolism and signaling pathways .

Which experimental models are most suitable for studying CAPRIN2 expression and function?

Several experimental models have proven effective for CAPRIN2 research:

Cell Culture Models:

• Neuronal cells: SH-SY5Y cells show robust CAPRIN2 expression

• Kidney cells: HEK-293 cells for Wnt signaling studies

• Ocular cells: Y79 cells for eye development research

• Chinese hamster ovary (CHO) cells: For translational regulation studies

Animal Models:

• Rat models: Particularly useful for hypothalamic studies involving osmotic regulation

• Mouse models: CAPRIN2 knockout mice show lens development defects

• Zebrafish: Effective for developmental studies, with CAPRIN2 morphants showing dorsalized phenotypes characteristic of Wnt pathway inhibition

Tissue Types:

• Brain tissue: Particularly hypothalamic nuclei (PVN and SON) for osmotic regulation studies

• Eye tissue: Especially lens fiber cells for differentiation studies

• Various tissues expressing Wnt pathway components

When selecting experimental models, consider tissue-specific expression patterns and the specific CAPRIN2 function under investigation. For osmotic regulation studies, hypothalamic nuclei provide the most relevant context, while lens tissue is optimal for studying differentiation mechanisms.

How should I select an appropriate CAPRIN2 antibody for my specific research application?

Selecting the optimal CAPRIN2 antibody requires careful consideration of several factors:

Target Species Reactivity:
Most commercial CAPRIN2 antibodies show reactivity with human, mouse, and rat samples . Verify sequence homology if studying other species.

Epitope Recognition:
Different antibodies target specific regions of CAPRIN2:

• Some antibodies target amino acids 88-329

• Others target regions within amino acids 500-650

Validation Data:
Review antibody validation data, particularly:

• Western blot showing a band of 126-150 kDa

• Knockdown/knockout controls confirming specificity

• Cross-reactivity testing with related proteins (e.g., CAPRIN1)

Application-Specific Considerations:

• For RNA immunoprecipitation studies, select antibodies validated for IP

• For co-localization studies in neurons, choose antibodies with demonstrated IF performance

• For quantitative Western blot analysis, select antibodies with linear signal response

For critical experiments, consider using multiple antibodies targeting different epitopes to confirm findings and include appropriate positive and negative controls in your experimental design.

How does CAPRIN2 regulate mRNA translation and what experimental approaches best reveal this mechanism?

CAPRIN2 functions as a translational regulator through several mechanisms that can be studied using specific experimental approaches:

Mechanism of Action:
CAPRIN2 binds to the translation initiation factor eIF3 and suppresses translation through inhibition of eIF3-dependent translation initiation . Ribosome profiling reveals that CAPRIN2 overexpression selectively reduces translation of long mRNAs, particularly those associated with cell proliferation, while shorter mRNAs escape this inhibition .

Experimental Approaches:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with anti-CAPRIN2 antibodies followed by Western blotting for eIF3 components

  • RNase A treatment to distinguish direct protein-protein interactions from RNA-mediated interactions

• Translational Efficiency Analysis:

  • Polysome profiling to examine global translation effects

  • Ribosome profiling to identify specifically affected mRNAs based on length and function

  • Puromycin incorporation assays to measure global protein synthesis rates

• RNA Binding Studies:

  • RNA immunoprecipitation to identify CAPRIN2-bound mRNAs

  • CLIP-seq for transcriptome-wide identification of binding sites

• Functional Validation:

  • Reporter assays comparing translation of long versus short reporter constructs

  • CAPRIN2 knockdown followed by analysis of protein synthesis rates

  • Analysis of 80S ribosome formation and elongation factor recruitment

This experimental framework can reveal how CAPRIN2 selectively regulates translation during cellular differentiation and stress responses, with important implications for understanding its role in both normal physiology and disease states.

What is CAPRIN2's role in the Wnt signaling pathway and how can this be experimentally investigated?

CAPRIN2 enhances canonical Wnt signaling through specific molecular interactions:

Mechanism:
CAPRIN2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3 (GSK3), enhancing the interaction between Axin and LRP5/6 . This interaction is critical for signal transmission from the plasma membrane to the cytosol.

Experimental Approaches:

• Protein Interaction Studies:

  • Co-immunoprecipitation of CAPRIN2 with LRP5/6

  • Western blotting for phosphorylated LRP5/6 to assess CAPRIN2's effect on receptor activation

  • Analysis of CAPRIN2-enhanced interaction between Axin and LRP5/6

• Signaling Pathway Analysis:

  • LEF-1/TCF reporter assays to measure Wnt pathway activation

  • Analysis of β-catenin stabilization in cytosolic fractions

  • qRT-PCR analysis of Wnt target gene expression

• Loss-of-Function Studies:

  • CAPRIN2 siRNA/shRNA knockdown followed by analysis of Wnt pathway activation

  • Rescue experiments with wildtype and truncated CAPRIN2 constructs

  • Domain mapping to identify regions required for Wnt pathway enhancement

• In Vivo Validation:

  • Zebrafish morpholino studies showing dorsalized phenotypes characteristic of Wnt inhibition

  • Rescue of CAPRIN2 morphant phenotypes by constitutively active β-catenin

These approaches collectively elucidate CAPRIN2's role in Wnt signaling, with implications for understanding its function in development and disease.

How does CAPRIN2 regulate AVP mRNA in the hypothalamus and what methodological approaches reveal this function?

CAPRIN2 binds directly to AVP mRNA in the hypothalamus, influencing water homeostasis through several mechanisms:

Mechanism:
In the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus, CAPRIN2 binds AVP mRNA and regulates its processing . This regulation is responsive to osmotic status, with salt loading and dehydration increasing CAPRIN2 expression.

Experimental Approaches:

• RNA Binding Analysis:

  • RNA immunoprecipitation (RIP) assays using anti-CAPRIN2 antibodies followed by qRT-PCR for AVP mRNA

  • Quantitative comparison with non-specific IgG controls (showing 23-108 fold enrichment)

  • Inclusion of control mRNAs (e.g., Rpl19) to confirm specificity

• In Vivo Functional Studies:

  • Stereotaxic delivery of lentiviral vectors expressing CAPRIN2 shRNA to the PVN and SON

  • Analysis of water balance parameters (urine output, fluid intake, urine osmolality)

  • Measurement of plasma AVP levels following CAPRIN2 knockdown

• Expression Analysis:

  • Quantification of AVP mRNA levels by qRT-PCR

  • Analysis of AVP protein expression by immunofluorescence

  • Correlation of CAPRIN2 expression with osmotic status

These methodologies collectively demonstrate CAPRIN2's critical role in regulating AVP mRNA processing and water homeostasis, providing insights into the central osmotic stress response.

What techniques are most effective for studying CAPRIN2's role in eye development and lens differentiation?

CAPRIN2 plays a crucial role in eye development, particularly in lens differentiation, which can be studied using the following approaches:

Mechanism:
CAPRIN2 regulates translation in lens cells, allowing shorter mRNAs (like crystallins) to escape translational inhibition while suppressing longer mRNAs associated with cell proliferation . This selective translational control is critical for proper lens fiber cell differentiation and nuclear compaction.

Experimental Approaches:

• In Vivo Developmental Analysis:

  • CAPRIN2 knockout or conditional knockout mouse models

  • Analysis of lens development using histological techniques

  • Measurement of lens nucleus size relative to entire lens

  • Wheat germ agglutinin (WGA) staining to visualize lens nucleus regions

• Molecular Mechanism Studies:

  • Ribosome profiling to identify translational effects on lens-specific mRNAs

  • Comparative analysis of short vs. long mRNA translation efficiency

  • Investigation of eIF3 interaction in lens tissue

• Cellular Differentiation Analysis:

  • Immunohistochemistry for crystallin expression and other differentiation markers

  • Analysis of cell proliferation vs. differentiation balance

  • Assessment of nuclear compaction in lens fiber cells

• Functional Validation:

  • Lens-specific CAPRIN2 knockdown

  • Rescue experiments with wildtype and truncated CAPRIN2 constructs

  • Comparative phenotypic analysis with other lens development mutants

These approaches provide complementary insights into CAPRIN2's role in lens development, connecting its molecular function in translational regulation to specific developmental outcomes.

How do I optimize Western blotting protocols for CAPRIN2 detection?

CAPRIN2 is a high molecular weight protein (126-150 kDa) that requires specific optimization for reliable Western blot detection:

Sample Preparation:

• Use RIPA buffer with protease inhibitors for efficient extraction

• Include phosphatase inhibitors if studying phosphorylation states

• Sonicate briefly to shear DNA and reduce sample viscosity

• Heat samples at 95°C for 5 minutes in Laemmadi buffer with DTT

Gel Electrophoresis:

• Use 8% or 6-10% gradient gels for optimal resolution of high molecular weight proteins

• Load sufficient protein (30-50 μg total protein per lane)

• Include molecular weight markers spanning 100-200 kDa range

• Run gel at lower voltage (80-100V) for better resolution

Transfer Conditions:

• Use wet transfer systems for high molecular weight proteins

• Add 0.1% SDS to transfer buffer to facilitate movement of large proteins

• Transfer at lower voltage (30V) overnight at 4°C for efficient transfer

• Use PVDF membranes (0.45 μm pore size) for better protein retention

Antibody Incubation:

• Block in 5% non-fat milk or BSA in TBST for 1-2 hours

• Dilute primary antibodies in range of 1:500-1:1000

• Incubate with primary antibody overnight at 4°C

• Use validated CAPRIN2 antibodies (e.g., 20766-1-AP) with confirmed specificity

Detection and Visualization:

• Use high-sensitivity detection reagents for optimal signal

• Start with longer exposure times (1-5 minutes) if signal is weak

• Expected molecular weight is 126-150 kDa

Common Troubleshooting:

• Multiple bands: May indicate splice variants or post-translational modifications

• No signal: Increase antibody concentration or protein loading

• High background: Increase blocking time and washing steps

Including positive controls (cells with known CAPRIN2 expression) and negative controls (CAPRIN2 knockdown samples) helps validate specificity and optimize detection conditions.

What are the optimal conditions for RNA immunoprecipitation (RIP) with CAPRIN2 antibodies?

RNA immunoprecipitation (RIP) is critical for studying CAPRIN2's RNA binding properties, particularly its interaction with AVP mRNA in the hypothalamus :

Tissue/Cell Preparation:

• For hypothalamic nuclei, rapidly dissect PVN and SON regions on ice

• For cultured cells, harvest at 80-90% confluence

• Consider crosslinking with 1% formaldehyde (10 minutes at room temperature)

• Lyse in non-denaturing buffer with RNase inhibitors

Immunoprecipitation:

• Pre-clear lysates with protein A/G beads (1 hour at 4°C)

• Use 2-5 μg of CAPRIN2 antibody validated for IP applications

• Always perform parallel IgG control IPs for background assessment

• Incubate with antibodies overnight at 4°C with gentle rotation

• Add pre-washed protein A/G beads and incubate 2-4 hours at 4°C

• Wash extensively with buffers of increasing stringency

RNA Extraction and Analysis:

• Extract RNA directly from beads using TRIzol or specialized kits

• Treat with DNase to remove genomic DNA contamination

• Perform reverse transcription followed by qPCR for specific target RNAs

• Calculate enrichment by comparing levels in CAPRIN2 IP vs. IgG control IP

Controls and Validation:

• Input control (5-10% of starting material)

• IgG immunoprecipitation control

• RNase treatment control to confirm RNA-dependence

• CAPRIN2 Western blot to confirm successful immunoprecipitation

• Include non-target RNA controls (e.g., Rpl19)

Expected Results:
For AVP mRNA binding to CAPRIN2 in hypothalamic nuclei, enrichment levels of 23-108 fold compared to IgG controls have been reported , while binding to control mRNAs like Rpl19 should be negligible.

This technique provides direct evidence of CAPRIN2's RNA binding activity and specificity in physiologically relevant contexts.

How do I design and validate CAPRIN2 knockdown experiments?

Effective CAPRIN2 knockdown studies require careful design and rigorous validation:

Design Considerations:

• Knockdown Method Selection:

  • siRNA: For transient knockdown in cultured cells

  • shRNA: For stable knockdown or in vivo applications via lentiviral delivery

  • Design targeting sequences to affect all known CAPRIN2 transcript variants

• Control Design:

  • Scrambled sequence controls with similar GC content

  • Mock transfection/transduction controls

  • Include fluorescent markers (e.g., eGFP) to identify transduced cells

• Delivery Method:

  • In vitro: Lipofection or nucleofection for cultured cells

  • In vivo: Stereotaxic delivery of lentiviral vectors to specific brain regions

Validation Framework:

• Molecular Validation:

  • qRT-PCR to confirm reduction in CAPRIN2 mRNA levels

  • Western blotting to verify protein knockdown

  • Immunofluorescence to assess knockdown at cellular level

  • Quantify knockdown efficiency (aim for >70% reduction)

• Specificity Controls:

  • Use multiple siRNA/shRNA sequences targeting different regions

  • Rescue experiments with RNAi-resistant CAPRIN2 constructs

  • Test effects on related family members (e.g., CAPRIN1)

• Functional Validation:

  • Assess known CAPRIN2-dependent processes:

    • Wnt signaling activation (LEF reporter assays)

    • Water homeostasis parameters

    • Translational regulation

• In Vivo Validation:

  • For hypothalamic knockdown, measure:

    • Urine output and fluid intake

    • Urine and plasma osmolality

    • Plasma AVP levels

  • For developmental studies:

    • Morphological phenotypes

    • Tissue-specific differentiation markers

Example Validation Data:

This comprehensive validation approach ensures that observed phenotypes are specifically attributed to CAPRIN2 knockdown rather than off-target effects.

What immunofluorescence protocols are optimal for studying CAPRIN2 localization and co-localization?

Optimized immunofluorescence protocols for CAPRIN2 localization studies:

Sample Preparation:

• For Cultured Cells:

  • Grow cells on poly-L-lysine coated coverslips

  • Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100 (5-10 minutes)

• For Tissue Sections:

  • Fix tissues in 4% paraformaldehyde (24-48 hours)

  • Cryoprotect in sucrose gradients (15-30%)

  • Section at 10-20 μm thickness

  • Perform heat-mediated antigen retrieval with citrate buffer (pH 6.0)

Staining Protocol:

• Blocking:

  • 5-10% normal serum in PBS with 0.1% Tween-20 (1 hour)

  • Use serum from the species of secondary antibody

• Primary Antibody Incubation:

  • CAPRIN2 antibody dilutions: 1:20-1:200

  • For co-localization, combine with antibodies against target proteins:

    • AVP for hypothalamic studies

    • eIF3 components for translation studies

    • LRP5/6 for Wnt signaling studies

  • Incubate overnight at 4°C in humid chamber

• Secondary Antibody Incubation:

  • Use species-specific, fluorophore-conjugated antibodies

  • Typically 1:200-1:500 dilution

  • Incubate 1-2 hours at room temperature

  • Include DAPI/Hoechst for nuclear counterstaining

Co-localization Analysis:

• Image Acquisition:

  • Use confocal microscopy for optimal spatial resolution

  • Capture Z-stacks through entire cell/tissue depth

  • Maintain consistent exposure settings across samples

  • Include single-label controls to assess bleed-through

• Quantitative Analysis:

  • Pearson's correlation coefficient for overlap assessment

  • Mander's coefficients for co-occurrence quantification

  • Line scan analysis across subcellular compartments

  • Colocalized pixel maps for visual representation

Application-Specific Considerations:

Expected Results:
In hypothalamic neurons, CAPRIN2 should co-localize with AVP in magnocellular neurosecretory cells (MCNs) . In cell culture systems under stress conditions, CAPRIN2 may form cytoplasmic puncta consistent with stress granule localization .

This protocol framework enables detailed analysis of CAPRIN2's subcellular localization and protein interactions across different experimental contexts.

How can I investigate CAPRIN2's role in translational regulation during cellular stress responses?

CAPRIN2 plays a critical role in stress responses through translational regulation, which can be investigated using these approaches:

Experimental Framework:

Expected Results:
CAPRIN2 overexpression should suppress global translation, with more pronounced effects on longer mRNAs . Under stress conditions, CAPRIN2 may recruit specific mRNAs to stress granules, modulating their translation. Experimental manipulation of CAPRIN2 levels should alter cellular responses to stress, including survival rates and recovery kinetics.

This experimental framework connects CAPRIN2's molecular function in translational regulation to its physiological role in stress adaptation, providing insights into both normal cellular physiology and potential disease mechanisms.

What approaches can reveal CAPRIN2's potential roles in neurodegenerative disorders?

CAPRIN2's involvement in RNA metabolism and stress responses suggests potential roles in neurodegenerative disorders that can be investigated using these approaches:

Experimental Framework:

• Expression Analysis in Disease Models:

  • Quantify CAPRIN2 levels in neurodegenerative disease models

  • Compare expression in affected vs. unaffected brain regions

  • Analyze correlation between CAPRIN2 levels and disease progression

  • Examine subcellular localization changes in disease states

• Stress Granule Dynamics:

  • Investigate CAPRIN2 incorporation into pathological RNA granules

  • Co-localization with disease-associated proteins (TDP-43, FUS, tau)

  • Analysis of stress granule formation and clearance kinetics

  • Effect of CAPRIN2 manipulation on stress granule properties

• Translational Regulation in Neurodegeneration:

  • Identify CAPRIN2-regulated mRNAs in neuronal contexts

  • Examine translation of neuroprotective vs. neurotoxic proteins

  • Analyze long vs. short mRNA translation in disease contexts

  • Effect of CAPRIN2 manipulation on protein aggregation

• Functional Interventions:

  • CAPRIN2 knockdown/overexpression in neurodegeneration models

  • Assessment of neuronal survival, morphology, and function

  • Analysis of dendritic complexity and synaptic maintenance

  • Evaluation of cellular stress responses and proteostasis

Methodological Approaches:

• Primary neuronal cultures from disease model animals

• iPSC-derived neurons from patients with neurodegenerative diseases

• Brain organoids for 3D modeling of disease processes

• In vivo models with region-specific CAPRIN2 manipulation

CAPRIN2's role in regulating translation of long mRNAs is particularly relevant, as many neurodegenerative disease-associated proteins are encoded by long transcripts. Additionally, its function in dendritic and synaptic maintenance suggests potential involvement in synaptic pathology characteristic of neurodegenerative disorders .

This multifaceted approach can reveal how CAPRIN2 contributes to neurodegeneration mechanisms and potentially identify new therapeutic targets for intervention.

What quality control measures ensure reliable results in CAPRIN2 antibody-based research?

Implementing comprehensive quality control measures is essential for generating reliable CAPRIN2 research data:

Antibody Validation:

• Western blot validation confirming single band of appropriate molecular weight (126-150 kDa)

• Testing across multiple cell lines/tissues with known CAPRIN2 expression

• Genetic validation using CAPRIN2 knockdown/knockout samples

• Peptide competition assays to confirm epitope specificity

• Multiple antibody approach using antibodies targeting different CAPRIN2 epitopes

Experimental Controls:

• Positive controls: Tissues/cells with confirmed CAPRIN2 expression (brain, Y79 cells)

• Negative controls: CAPRIN2 knockdown/knockout samples, non-specific IgG for IP

• Technical replicates: Minimum of three independent experiments

• Biological replicates: Multiple samples from different sources

• Dose-response relationships when manipulating CAPRIN2 levels

Data Analysis Quality Controls:

• Blinded analysis where possible to prevent observer bias

• Appropriate statistical methods for data type and distribution

• Multiple analytical approaches to confirm key findings

• Clear documentation of all analysis parameters and exclusion criteria

Reproducibility Measures:

• Detailed protocol documentation including all buffer compositions

• Reporting of antibody catalog numbers, lot numbers, and dilutions

• Standardized sample preparation and handling procedures

• Consistent imaging parameters for microscopy-based experiments

Application-Specific Controls:

Implementing these quality control measures ensures that findings are specifically attributable to CAPRIN2 and facilitates reproducibility across different research laboratories and experimental systems.

How can researchers integrate multiple techniques to comprehensively characterize CAPRIN2 function in their specific research context?

A comprehensive understanding of CAPRIN2 function requires integration of multiple complementary techniques:

Multidisciplinary Experimental Framework:

• Expression Analysis:

  • qRT-PCR for mRNA quantification

  • Western blotting for protein levels

  • Immunohistochemistry for tissue localization

  • RNA-seq for transcriptomic context

• Functional Manipulation:

  • RNAi-mediated knockdown

  • CRISPR/Cas9 gene editing for knockout models

  • Overexpression studies with wildtype and mutant constructs

  • Domain-specific deletion constructs

• Interaction Studies:

  • RNA immunoprecipitation for RNA binding partners

  • Co-immunoprecipitation for protein interactions

  • Proximity ligation assays for in situ interaction detection

  • Mass spectrometry for unbiased interactome analysis

• Functional Readouts:

  • Translational efficiency measurements

  • Signaling pathway activation assays

  • Physiological parameters in animal models

  • Cell type-specific phenotypic analyses

Integration Strategies:

• Sequential Investigation:
  Begin with expression analysis to establish context, followed by knockdown studies to assess function, interaction studies to determine mechanisms, and functional readouts to confirm physiological relevance.

• Parallel Approaches:
  Simultaneously employ multiple techniques (e.g., combining RNA-IP with ribosome profiling) to capture different aspects of CAPRIN2 function within the same experimental system.

• Cross-Validation:
  Confirm key findings using alternative technical approaches (e.g., validate protein interactions identified by mass spectrometry using co-immunoprecipitation).

• Spatial and Temporal Integration:
  Analyze CAPRIN2 function across different cellular compartments, developmental stages, and physiological conditions to build a comprehensive functional map.

Contextual Application:
Tailor this integrated approach to specific research questions:

This multifaceted approach provides robust, convergent evidence for CAPRIN2 function, overcoming limitations of individual techniques and revealing emergent properties that might be missed by more narrowly focused studies.