reep3 Antibody

What is REEP3 and what are its primary cellular functions?

REEP3 (Receptor Expression-Enhancing Protein 3) is a transmembrane protein with several critical cellular functions. Initially discovered for its interaction with cell surface receptors, REEP3 plays a role in enhancing receptor expression and stability . It serves as a pivotal enzyme crucial for endoplasmic reticulum (ER) clearance during mitosis . Additionally, REEP3 assists in shaping the endoplasmic reticulum by facilitating the stacking and proper organization of ER membranes .

As a microtubule-binding protein, REEP3 is required to ensure proper cell division and nuclear envelope reassembly, specifically by sequestering the endoplasmic reticulum away from chromosomes during mitosis . This multi-functional nature positions REEP3 at the intersection of cellular architecture, division, and signaling pathways. Recent research has also implicated REEP3 in diverse pathological conditions, including pancreatic cancer and neurodevelopmental disorders .

How are REEP3 antibodies typically validated for research applications?

Validating REEP3 antibodies requires a multi-faceted approach to ensure specificity and reliability in research applications. The most robust validation strategy includes:

• Knockout/knockdown controls: Using CRISPR-Cas9 or siRNA to deplete REEP3 and confirm antibody signal reduction.

• Immunoprecipitation validation: As demonstrated with antibodies like ab241964, REEP3 antibodies can be validated through immunoprecipitation from cell lysates (e.g., HeLa cells), with typical protocols using 8 μl of antibody per reaction for IP and 1:400 dilution for subsequent Western blot detection .

• Cross-reactivity assessment: Testing against related REEP family proteins (REEP1, REEP2, REEP4, REEP5, and REEP6) to confirm specificity for REEP3.

• Multiple detection methods: Confirming consistent results across different applications such as Western blotting, immunofluorescence, and ELISA .

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide to confirm specific binding is blocked.

Researchers should thoroughly document these validation steps as part of their experimental protocols to ensure reproducibility and reliability of results.

What cell lines are recommended for REEP3 expression studies?

Based on current research findings, several cell lines are particularly suitable for REEP3 expression studies:

• HeLa cells: Human cervical adenocarcinoma cell line with demonstrated REEP3 expression, successfully used in immunoprecipitation studies and validated for antibody testing .

• Pancreatic cancer cell lines: Given the significant upregulation of REEP3 in pancreatic cancer tissues, these cell lines provide an excellent model for studying REEP3 overexpression and its functional consequences .

• Neural cell lines: Since REEP3 has been suggested as a candidate gene for autism, neuronal cell lines are valuable for investigating its neural functions and potential role in neurodevelopmental disorders .

When selecting cell lines, researchers should prioritize those that naturally express REEP3 at detectable levels or are relevant to the specific biological context being studied, such as cancer research or neurodevelopmental studies.

What is the recommended protocol for REEP3 immunoprecipitation?

An optimized protocol for REEP3 immunoprecipitation, based on validated research methods, includes:

• Cell lysate preparation:

  • Prepare whole cell lysate using NETN lysis buffer (typically containing NaCl, EDTA, Tris-HCl, and NP-40)

  • Use approximately 1 mg of protein per IP reaction

  • Ensure complete cell lysis through gentle mechanical disruption

• Immunoprecipitation procedure:

  • Add REEP3 antibody (e.g., ab241964) at approximately 8 μl per reaction

  • Incubate with protein lysate (4°C overnight with gentle rotation)

  • Add protein A/G beads and continue incubation (2-4 hours)

  • Perform 3-5 wash steps with NETN buffer

  • Elute precipitated proteins with appropriate sample buffer

• Analysis:

  • Load approximately 20% of IP sample for Western blot verification

  • Use the same REEP3 antibody at 1:400 dilution for detection

  • Include control IgG lane for comparison

  • Visualize using chemiluminescence (30 seconds exposure is typically sufficient)

This protocol has been validated for human cell lines like HeLa. Researchers should optimize antibody amounts and incubation conditions for different sample types.

How do different applications affect REEP3 antibody performance?

REEP3 antibodies demonstrate varying performance across different experimental applications, and researchers should optimize conditions specifically for their application of interest:

• Immunoprecipitation (IP): REEP3 antibodies such as ab241964 have demonstrated strong performance in IP applications, successfully pulling down REEP3 from cell lysates. The recommended concentration is approximately 8 μl per reaction .

• Western Blotting (WB): For effective detection in Western blots, antibodies are typically used at dilutions around 1:400. Proper sample preparation is crucial, as is the inclusion of appropriate molecular weight markers to confirm the expected band size for REEP3 .

• ELISA: Certain REEP3 antibodies are specifically developed for ELISA applications, allowing quantitative detection in various sample types .

• Immunohistochemistry/Immunofluorescence: While optimization is required, REEP3 antibodies can be adapted for these applications based on epitope accessibility in fixed tissues or cells.

Performance optimization typically requires proper sample preparation, appropriate antibody concentration, and validated detection methods. Researchers should initially test multiple conditions to determine optimal parameters for their specific experimental system.

What are the best fixation methods for REEP3 immunostaining?

Optimal fixation methods for REEP3 immunostaining should consider its dual nature as both a membrane-associated protein and an ER-shaping protein:

• Paraformaldehyde fixation:

  • 4% paraformaldehyde for 10-15 minutes at room temperature

  • This preserves protein structure while maintaining epitope accessibility

  • Particularly suitable for REEP3 as it maintains membrane protein conformation

• Methanol fixation:

  • 100% methanol at -20°C for 10 minutes

  • Effective for preserving protein-protein interactions

  • May better expose intracellular epitopes of REEP3

• Hybrid protocols:

  • Initial fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100

  • This combined approach preserves both membrane structure and enables antibody access to intracellular domains of REEP3

Since REEP3 is involved in both ER shaping and microtubule binding , the optimal fixation method may depend on which aspect of REEP3 biology is being investigated. Empirical testing with different fixation protocols is recommended for each specific REEP3 antibody to ensure epitope preservation and accessibility.

How can REEP3 antibodies be used to study its role in mitosis and nuclear envelope dynamics?

REEP3 antibodies can be employed in sophisticated experimental approaches to investigate its role in mitosis and nuclear envelope dynamics:

• Immunofluorescence microscopy during cell cycle progression:

  • Using synchronized cell populations fixed at specific mitotic stages

  • Co-staining with markers for chromosomes (DAPI), microtubules (α-tubulin), and nuclear envelope components

  • This approach can visualize how REEP3 clears ER membranes from metaphase chromosomes

• Super-resolution microscopy techniques:

  • STORM, PALM, or SIM to visualize REEP3-dependent ER clearing around chromosomes at nanoscale resolution

  • Multi-color imaging to observe spatial relationships between REEP3, microtubules, and chromosomes

• Proximity labeling combined with antibody detection:

  • APEX2 or BioID fused to REEP3 to identify proximal proteins during mitosis

  • Validation of identified interactions using co-immunoprecipitation with REEP3 antibodies

  • This approach identifies novel REEP3 interaction partners specific to mitotic processes

• Live-cell imaging with antibody fragments:

  • Anti-REEP3 antibody fragments (Fab) conjugated to cell-permeable fluorophores

  • Development of antibody-based FRET sensors to detect REEP3 conformational changes during mitosis

These methodologies, particularly when used in combination, provide comprehensive insights into REEP3's mechanistic role in coordinating ER dynamics and nuclear envelope reassembly during cell division.

What is the relationship between REEP3 expression and cancer progression?

REEP3 expression shows significant alterations across cancer types, with particularly notable patterns in pancreatic cancer that suggest its potential as both a diagnostic and prognostic marker:

These alterations position REEP3 as both a potential diagnostic biomarker and prognostic indicator in cancer research, particularly for pancreatic cancer.

How does REEP3 interact with the immune microenvironment in cancer?

REEP3 demonstrates significant interactions with the immune microenvironment in cancer, suggesting potential implications for immunotherapy research:

• Correlation with immune cell infiltration:

  • REEP3 expression shows significant positive correlations with multiple immune cell types:

    • B cells (r = 0.201)

    • CD8+ T cells

    • Dendritic cells

    • Macrophages

    • Neutrophils

• Impact of REEP3 copy number variation (CNV):

  • REEP3 CNV significantly correlates with infiltration levels of:

    • B cells

    • CD4+ T cells

    • Macrophages

    • Neutrophils

• Differential immune cell profiles based on REEP3 expression:

  • High REEP3 expression correlates with increased infiltration of:

    • Activated B cells and T cells (CD4+ and CD8+)

    • Gamma delta T cells

    • Type 1 and Type 2 T helper cells

    • Activated dendritic cells

    • Natural killer cells and macrophages

• Relationship with immune checkpoint markers:

  • Significant differences in expression of immune checkpoint markers between high and low REEP3 expression groups, including:

    • CD274 (PD-L1)

    • CD47

    • CD80

    • LAG3

    • PVR

    • TNFRSF18

    • TNFSF4

These findings suggest REEP3 may play a significant role in shaping the tumor immune microenvironment, offering potential applications in immunotherapy research and development of combination therapeutic approaches.

What are the emerging applications of REEP3 antibodies in neurodevelopmental disorder research?

REEP3 antibodies are finding emerging applications in neurodevelopmental disorder research, particularly in autism spectrum disorders:

• Genetic association validation:

  • REEP3 has been suggested as a novel candidate gene for autism

  • Antibodies can validate expression differences in autism-relevant neural tissues

  • Immunohistochemistry or immunoblotting on neural samples can provide protein-level validation of genetic findings

• Receptor trafficking investigations:

  • Given REEP3's function in enhancing receptor expression , antibodies can track:

    • Co-localization with neurodevelopmentally relevant receptors

    • Alterations in receptor trafficking in autism models

    • Changes in REEP3-receptor interactions during neural development

• Developmental expression profiling:

  • Immunohistochemistry to map REEP3 expression through neurodevelopmental stages

  • Comparison between typically developing brains and autism models

  • Correlation of expression patterns with critical periods of circuit formation

• Functional studies in iPSC-derived neurons:

  • REEP3 antibodies to assess expression in induced pluripotent stem cell (iPSC)-derived neurons from:

    • Neurotypical individuals

    • Individuals with autism carrying REEP3 variants

• Biomarker exploration:

  • Given REEP3's influence on alkaline phosphatase levels , antibody-based detection in samples could potentially correlate with neurodevelopmental profiles

These applications leverage REEP3 antibodies to bridge genetic associations with functional consequences in neurodevelopmental disorders, potentially identifying new therapeutic targets or diagnostic approaches.

What signaling pathways are associated with REEP3 expression?

Functional enrichment analyses have revealed multiple signaling pathways significantly associated with REEP3, particularly in the context of pancreatic cancer:

Additionally, Gene Ontology (GO) analyses have demonstrated enrichment in biological processes including cytoplasmic translation, wound healing, viral processes, regulation of cellular component size, and actin filament organization . In terms of cellular components, REEP3 is associated with cell-substrate junctions, focal adhesions, ribosomes, and cytosolic components .

These pathway associations provide important insights for researchers investigating REEP3's functional role in both normal and pathological contexts.

How does REEP3 expression vary across tissue types and disease states?

REEP3 expression demonstrates significant variation across tissue types and disease states, particularly in the context of cancer:

This expression profile has been validated across multiple datasets including TCGA, GTEx, and GEO cohorts . The consistent upregulation in cancer tissues compared to normal tissues, along with the impressive diagnostic value (AUC) across different cancer stages, highlights REEP3's potential utility as a biomarker.

How do REEP3 antibodies compare in performance across different applications?

While comprehensive comparative data on different REEP3 antibodies is limited in the literature, available information suggests varying performance characteristics across antibodies and applications:

Performance notes from available studies:

• For immunoprecipitation: 8 μl per reaction of ab241964 has been validated

• For Western blotting: 1:400 dilution is reported as effective

• For detection: Chemiluminescence with 30 seconds exposure time is sufficient with some antibodies

Researchers should conduct their own comparative analyses when selecting antibodies for specific applications, as performance can vary significantly depending on the experimental context, sample type, and detection method.

What are the challenges in targeting REEP3 for therapeutic applications?

Targeting REEP3 for therapeutic applications presents several significant challenges that researchers must address:

• Functional redundancy within the REEP family:

  • REEP3 belongs to a family that includes REEP1-6

  • Close relationships with REEP1, REEP2, and REEP4 may lead to compensatory mechanisms if only REEP3 is targeted

• Essential cellular functions:

  • REEP3's role in fundamental processes like ER organization and cell division means inhibition could cause significant off-target effects

  • Complete inhibition might result in cellular toxicity due to disruption of critical mitotic processes

• Complex signaling pathway involvement:

  • REEP3's implication in multiple signaling pathways (TGF-beta, AR pathway, ERBB1 downstream pathway) makes selective pathway inhibition challenging

• Dual role in immune modulation:

  • REEP3's positive correlation with immune cell infiltration suggests it may have both pro- and anti-tumor effects depending on context

  • Targeting strategies need to consider potential effects on anti-tumor immunity

• Antibody accessibility limitations:

  • As a transmembrane protein, developing antibodies that can access functionally important domains may be technically challenging

  • Limited accessibility to certain epitopes in live cells or in vivo

Addressing these challenges requires multi-faceted approaches, possibly combining partial REEP3 inhibition with complementary therapeutic strategies that target associated pathways or immune components.

How can researchers address conflicting data regarding REEP3 function?

Researchers can systematically address conflicting data regarding REEP3 function through several methodological approaches:

• Standardization of detection methods:

  • Use multiple validated antibodies targeting different REEP3 epitopes

  • Implement quantitative approaches like qPCR for mRNA and quantitative western blotting for protein

  • Include appropriate positive and negative controls in all experiments

• Context-specific analysis:

  • Evaluate REEP3 expression and function across different cell types, tissues, developmental stages, and disease states

  • This can reveal whether conflicts arise from biological context differences rather than methodological issues

• Integrated multi-omics approach:

  • Combine transcriptomics, proteomics, and functional assays

  • Correlate REEP3 expression with pathway activation markers

  • Implement single-cell analyses to address heterogeneity within populations

• Genetic approaches:

  • Implement CRISPR-Cas9 knockout/knockin studies

  • Use rescue experiments with wild-type and mutant REEP3

  • Apply inducible expression systems to study acute vs. chronic effects

• Consideration of REEP family redundancy:

  • Assess compensation by other REEP family members (REEP1, REEP2, REEP4, REEP5, REEP6)

  • Implement combinatorial knockdown/knockout approaches

  • Evaluate cross-reactivity of detection methods with other REEP proteins

By systematically addressing conflicts through these approaches, researchers can develop a more nuanced and accurate understanding of REEP3 biology across different contexts.