REPS2 Antibody

What is REPS2 and what cellular functions does it regulate?

REPS2 (RALBP1 Associated Eps Domain Containing Protein 2), also known as POB1, is a multifunctional protein involved in growth factor signaling through its influence on the Ral signaling pathway . It plays a critical role in ligand-dependent receptor-mediated endocytosis of epidermal growth factor (EGF) and insulin receptors . REPS2 functions by binding to RALBP1, thereby inhibiting the RALBP1/RAL signaling pathway, which consequently inhibits the endocytosis of EGF and insulin . By controlling growth factor receptor endocytosis, REPS2 regulates cell survival and, through ASAP1, may modulate cell adhesion and migration .

Recent research has implicated REPS2 in various cancer types. Downregulation of REPS2 expression coincides with the androgen-dependent to androgen-independent transition in prostate cancer . REPS2 also binds to p65 reciprocally to inhibit NF-κB activity and is involved in the development and prognosis of breast cancer, esophageal squamous cell carcinoma, and colorectal cancer .

What applications are REPS2 antibodies suitable for?

REPS2 antibodies have been validated for multiple research applications. The table below summarizes available applications by antibody format:

When selecting an antibody for your research, consider the specific application requirements, target species, and whether polyclonal or monoclonal antibodies would be more appropriate for your experimental design .

What is the expected molecular weight of REPS2 in Western blot analysis?

• 72 kDa (full-length protein)

• 55 kDa (potential isoform or post-translationally modified variant)

• 30 kDa (potential degradation product or processed form)

These variations in observed molecular weight may depend on cell type, experimental conditions, and potential post-translational modifications. When validating your REPS2 antibody, it is advisable to use positive control lysates from cells known to express REPS2, such as LnCap, T47-D, or 293T cells, which have been documented to express detectable levels of REPS2 .

How does REPS2 regulate receptor-mediated endocytosis?

REPS2 plays a crucial role in the endocytosis of growth factor receptors through multiple mechanisms:

• RALBP1 Interaction: REPS2 binds to RALBP1, inhibiting the RALBP1/RAL signaling pathway that normally promotes receptor endocytosis .

• EGF-mediated EGFR Endocytosis: Research demonstrates that REPS2 inhibits EGF-mediated endocytosis of EGFR as well as downstream signaling pathways including AKT/NF-κB, p38MAPK, and ERK1/2 .

• Cell Survival Regulation: By controlling growth factor receptor endocytosis, REPS2 may regulate cell survival mechanisms .

• Cell Adhesion and Migration: Through interaction with ASAP1, REPS2 may regulate cell adhesion and migration processes .

In hepatocellular carcinoma (HCC) cells, increased expression of REPS2 (induced by T317, a Liver X Receptor agonist) inhibits EGF-mediated endocytosis of EGFR and subsequently downregulates the activation of downstream signaling pathways . This regulatory mechanism provides potential therapeutic targets for cancer treatment, as REPS2 expression levels are inversely correlated with HCC development, with reduced expression associated with poor prognosis .

What is known about REPS2 expression in cancer and its prognostic significance?

REPS2 has emerged as a significant player in cancer biology with distinct expression patterns and prognostic implications:

• Hepatocellular Carcinoma (HCC): Clinical data analysis reveals that REPS2 expression levels are inversely correlated with HCC development. Reduced REPS2 expression is associated with poor prognosis, suggesting that REPS2 might function as a tumor suppressor in HCC .

• Prostate Cancer: Downregulation of REPS2 expression coincides with the transition from androgen-dependent to androgen-independent prostate cancer, indicating a potential role in disease progression .

• Other Cancers: REPS2 is also involved in the development and prognosis of breast cancer, esophageal squamous cell carcinoma, and colorectal cancer, though the specific mechanisms may vary across cancer types .

Mechanistically, REPS2 exerts its tumor-suppressive effects by:

• Binding to p65 reciprocally to inhibit NF-κB activity

• Inhibiting EGF-mediated endocytosis of EGFR and downstream signaling pathways including AKT/NF-κB, p38MAPK, and ERK1/2

• Potentially modulating cell adhesion and migration through interaction with ASAP1

These findings suggest that REPS2 could serve as a prognostic biomarker and potential therapeutic target in multiple cancer types.

What are the key considerations when designing immunoprecipitation experiments with REPS2 antibodies?

When designing immunoprecipitation (IP) experiments with REPS2 antibodies, researchers should consider several key factors:

• Antibody Selection: Recombinant monoclonal antibodies such as EPR12033(B) (ab170883 and ab249563) have been specifically validated for IP applications with human samples . These antibodies have demonstrated efficacy in precipitating REPS2 from cell lysates.

• Cell Line Selection: 293T cells have been documented as effective sources for REPS2 immunoprecipitation . Other cell lines with high REPS2 expression, such as LnCap and T47-D, may also be suitable.

• Experimental Protocol:

  • Antibody concentration: Start with a 1/10 dilution of the REPS2 antibody for immunoprecipitation

  • For Western blot detection of immunoprecipitated REPS2, use a 1/10,000 dilution of the same antibody

  • Ensure proper controls are included (IgG control, input control)

• Validation: Confirm the specificity of the immunoprecipitation by:

  • Western blot analysis of the IP pellet

  • Comparing with known molecular weights (expected 72 kDa for full-length REPS2)

  • Including appropriate negative controls

• Downstream Applications: Consider how the immunoprecipitated REPS2 will be used (protein-protein interaction studies, post-translational modification analysis, etc.) and adjust your protocol accordingly.

By carefully considering these factors, researchers can optimize their REPS2 immunoprecipitation experiments for successful outcomes and reliable data.

How is REPS2 expression regulated at the transcriptional level?

REPS2 expression is regulated through several transcriptional mechanisms, with recent research highlighting the role of Liver X Receptors (LXRs):

• LXR-mediated Regulation: Studies have demonstrated that LXRα/β play a critical role in regulating REPS2 expression. Knockdown of LXRα/β in HepG2 cells significantly decreases REPS2 expression .

• LXRE in REPS2 Promoter: A functional LXR-response element (LXRE) has been identified in the REPS2 promoter region. The binding of LXR protein to this LXRE enhances REPS2 transcription .

• T317 Compound Effects: The LXR agonist T317 enhances REPS2 expression at the transcriptional level by promoting the binding of LXR protein to the LXRE in the REPS2 promoter region .

The specific methodological approach to study this regulation includes:

• Promoter Activity Assay: The human REPS2 promoter (from −1687 to −921) can be amplified using PCR and inserted into a luciferase reporter vector (pGL4.10) to study promoter activity .

• Site-directed Mutagenesis: Mutation of the predicted LXRE sequence allows for functional validation of the LXR binding site .

• Chromatin Immunoprecipitation (ChIP) Assay: To determine the binding of LXR protein to the LXRE in the REPS2 promoter, researchers treat cells with T317 (400 nM) for 18 hours, then perform ChIP using specific LXRα or LXRβ monoclonal antibodies .

• ChIP Primers: Forward 5′-TCAAGCCTGTAATCCCAGCACTTT-3′, Reverse 5′-GGCTGTAGTTCAATGGCACAGTCTT-3′ .

This transcriptional regulation of REPS2 represents a potential therapeutic target, particularly in cancer contexts where REPS2 expression is frequently dysregulated.

What methodological approaches can be used to study REPS2 function in receptor endocytosis pathways?

To investigate REPS2's role in receptor endocytosis pathways, researchers can employ several sophisticated methodological approaches:

• Gene Expression Modulation:

  • RNA interference (siRNA or shRNA) targeting REPS2

  • CRISPR-Cas9 gene editing to create REPS2 knockout models

  • Overexpression systems using plasmid vectors containing REPS2 cDNA

• Receptor Internalization Assays:

  • Fluorescently labeled EGF or insulin to track receptor internalization

  • Flow cytometry to quantify surface receptor expression before and after ligand stimulation

  • Confocal microscopy with dual-labeled antibodies to simultaneously track REPS2 and receptor localization

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation using REPS2 antibodies to identify binding partners

  • Proximity ligation assays to visualize protein interactions in situ

  • FRET/BRET assays to study dynamic protein interactions

• Signaling Pathway Analysis:

  • Phospho-specific antibodies to monitor activation of downstream pathways (AKT/NF-κB, p38MAPK, ERK1/2) following EGF stimulation in the presence and absence of REPS2

  • Inhibitors of specific pathway components to dissect the hierarchy of signaling events

  • Time-course experiments to determine the kinetics of receptor internalization and signal transduction

• Live Cell Imaging:

  • REPS2-GFP fusion proteins to track localization during endocytosis

  • TIRF microscopy to visualize membrane-proximal events

  • High-content imaging to quantify endocytic vesicle formation

These methodological approaches can be combined to provide a comprehensive understanding of how REPS2 regulates receptor endocytosis and influences downstream signaling pathways in normal and pathological conditions.

What are the challenges and solutions for validating REPS2 antibody specificity?

Validating antibody specificity is crucial for generating reliable research data. For REPS2 antibodies, researchers face several challenges and can implement specific solutions:

Challenges in REPS2 Antibody Validation:

• Multiple Observed Molecular Weights: REPS2 can be detected at 72 kDa (full-length), 55 kDa, and 30 kDa, complicating interpretation of Western blot results .

• Cross-Reactivity Concerns: Some antibodies show reactivity with both human and mouse REPS2, requiring careful validation across species .

• Isoform Recognition: Multiple isoforms or post-translationally modified forms may exist, affecting antibody recognition.

• Background Signal: Non-specific binding can complicate analysis, particularly in techniques like immunohistochemistry.

Methodological Solutions:

• Genetic Validation:

  • Use REPS2 knockout or knockdown models as negative controls

  • Compare antibody signal in cells with varying REPS2 expression levels

  • Employ CRISPR-Cas9 gene editing to create epitope-tagged REPS2 for validation

• Multiple Antibody Approach:

  • Use antibodies targeting different epitopes (N-terminal vs. internal regions)

  • Compare polyclonal antibodies (broader epitope recognition) with monoclonal antibodies (higher specificity)

  • Validate findings with both rabbit and mouse host antibodies

• Application-Specific Validation:

  • For Western blotting: Include positive controls (LnCap, T47-D, or 293T cell lysates)

  • For immunoprecipitation: Confirm pulled-down protein by mass spectrometry

  • For immunofluorescence: Include peptide competition assays

• Quantitative Validation:

  • Titrate antibody concentrations (1:10,000 for WB, 1:10 for IP)

  • Include loading controls and quantify relative expression

  • Perform dose-response relationships with REPS2-inducing agents like T317

By implementing these methodological approaches, researchers can ensure the specificity and reliability of their REPS2 antibodies across multiple experimental applications.

How does REPS2 interact with the EGFR signaling pathway, and what methods can detect these interactions?

REPS2 plays a critical regulatory role in EGFR signaling through several mechanisms that can be studied using specific methodological approaches:

REPS2-EGFR Signaling Interactions:

• Inhibition of EGFR Endocytosis: REPS2 inhibits EGF-mediated endocytosis of EGFR, affecting receptor turnover and sustained signaling .

• Downstream Pathway Regulation: REPS2 modulates the activation of multiple EGFR-dependent signaling cascades:

  • AKT/NF-κB pathway

  • p38MAPK pathway

  • ERK1/2 signaling pathway

• RALBP1 Pathway Interaction: REPS2 binds to RALBP1, inhibiting the RALBP1/RAL signaling pathway that normally promotes receptor endocytosis .

Methodological Approaches to Study These Interactions:

• Receptor Internalization Assays:

  • Biotinylation of cell surface proteins followed by internalization assays

  • Flow cytometry with fluorescently labeled anti-EGFR antibodies

  • Pulse-chase experiments with labeled EGF

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation using antibodies against REPS2 (such as EPR12033(B))

  • Proximity ligation assay to visualize REPS2-EGFR interactions in situ

  • Pull-down assays with recombinant REPS2 domains

• Signaling Pathway Analysis:

  • Western blotting with phospho-specific antibodies targeting:

    • p-EGFR (Y1068, Y1173)

    • p-AKT (S473, T308)

    • p-ERK1/2 (T202/Y204)

    • p-p38MAPK (T180/Y182)

  • Kinetic studies to determine temporal dynamics of pathway activation

  • Pathway inhibitor studies to establish signaling hierarchies

• Transcriptional Regulation Analysis:

  • ChIP assays to study NF-κB binding to target genes

  • Reporter gene assays for NF-κB-responsive elements

  • RT-qPCR of EGFR pathway target genes

• Functional Outcome Measurements:

  • Cell proliferation assays (e.g., MTT, BrdU incorporation)

  • Migration assays (wound healing, transwell)

  • Apoptosis assays (Annexin V/PI staining, caspase activity)

By integrating these methodological approaches, researchers can comprehensively characterize how REPS2 modulates EGFR signaling in normal and pathological contexts, such as cancer development where REPS2 expression is frequently altered .

What are the optimal storage and handling conditions for REPS2 antibodies?

For maximum stability and performance of REPS2 antibodies, researchers should follow these storage and handling recommendations:

• Storage Temperature:

  • Store antibodies at -20°C for long-term stability

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

• Buffer Composition:

  • REPS2 antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some formulations may contain 0.1% BSA for additional stability

• Stability Information:

  • Most REPS2 antibodies remain stable for one year after shipment when stored properly

  • For smaller size (20μl) antibody preparations containing BSA, special storage considerations may apply

• Handling Practices:

  • Thaw antibodies on ice before use

  • Centrifuge briefly to collect contents at the bottom of the tube

  • Avoid contamination by using sterile technique

  • Return to -20°C storage promptly after use

• Working Dilution Preparation:

  • Prepare fresh working dilutions on the day of use

  • For Western blotting, a 1:10,000 dilution is recommended for some REPS2 antibodies

  • For immunoprecipitation, a 1:10 dilution has been validated

Following these storage and handling guidelines will help ensure consistent performance and reliable results when using REPS2 antibodies in various experimental applications.

How can researchers optimize REPS2 antibodies for use in immunofluorescence studies?

Optimizing immunofluorescence (IF) protocols for REPS2 detection requires careful consideration of several methodological parameters:

• Antibody Selection:

  • Choose antibodies specifically validated for IF applications (e.g., 20699-1-AP, ABIN7136253)

  • Consider using antibodies targeting different epitopes for confirmation

• Fixation Method Optimization:

  • Compare paraformaldehyde (4%, 10-15 minutes) with methanol fixation (-20°C, 10 minutes)

  • For membrane-associated REPS2 detection, mild fixation may better preserve epitopes

  • Consider dual fixation (paraformaldehyde followed by methanol) for detecting both membrane and cytoplasmic pools

• Permeabilization Conditions:

  • Test different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.5% saponin)

  • Optimize permeabilization time (5-15 minutes) to balance antibody access with structural preservation

• Blocking Strategy:

  • Use 5-10% normal serum (from the species of the secondary antibody)

  • Add 1% BSA to reduce non-specific binding

  • Consider adding 0.1-0.3% Triton X-100 in blocking buffer for better penetration

• Antibody Dilution Titration:

  • Start with manufacturer's recommended dilution and test a series of dilutions

  • Include a negative control (no primary antibody) to assess background

• Signal Amplification Methods:

  • For weak signals, consider tyramide signal amplification

  • Evaluate different fluorophore-conjugated secondary antibodies (Alexa Fluor vs. DyLight)

• Co-localization Studies:

  • For REPS2 and EGFR co-localization, use species-distinct primary antibodies

  • Include appropriate controls for spectral bleed-through

  • Analyze with quantitative co-localization software (e.g., JACoP plugin for ImageJ)

• Confocal Imaging Parameters:

  • Use sequential scanning to minimize cross-talk between channels

  • Optimize pinhole size, gain, and laser power to maximize signal-to-noise ratio

  • Consider deconvolution for improved resolution

By systematically optimizing these parameters, researchers can achieve specific and sensitive detection of REPS2 in immunofluorescence studies, enabling detailed analysis of its subcellular localization and co-localization with interaction partners.

How can REPS2 antibodies be utilized in cancer research and potential therapeutic development?

REPS2 antibodies offer several valuable applications in cancer research and therapeutic development:

• Prognostic Biomarker Validation:

  • REPS2 expression is inversely correlated with HCC development and prognosis

  • Immunohistochemistry with validated REPS2 antibodies can help stratify patients and predict outcomes

  • Tissue microarray analysis can establish correlations between REPS2 expression and clinical parameters

• Therapeutic Target Assessment:

  • Monitoring REPS2 expression changes in response to LXR agonists like T317

  • Evaluating effects of REPS2 modulation on cancer cell proliferation and migration

  • Screening for compounds that enhance REPS2 expression or activity

• Mechanism of Action Studies:

  • Investigating how REPS2 regulates EGFR endocytosis in different cancer types

  • Examining the interplay between REPS2 and NF-κB signaling in tumor progression

  • Studying REPS2's role in the transition from androgen-dependent to androgen-independent prostate cancer

• Combination Therapy Research:

  • Evaluating synergistic effects between REPS2-targeting approaches and conventional therapies

  • Identifying resistance mechanisms by analyzing REPS2 expression patterns

• Methodological Approaches:

  • Use immunoprecipitation with monoclonal REPS2 antibodies followed by mass spectrometry to identify novel interaction partners

  • Employ tissue microarrays with REPS2 antibodies to correlate expression with patient outcomes

  • Develop phospho-specific REPS2 antibodies to study its post-translational regulation

• Translational Applications:

  • Design companion diagnostic tests using REPS2 antibodies to guide targeted therapy selection

  • Develop antibody-drug conjugates targeting REPS2-expressing cells

  • Create imaging agents based on REPS2 antibodies for cancer detection

By leveraging these applications, researchers can advance our understanding of REPS2's role in cancer and potentially develop novel therapeutic strategies targeting this protein or its regulatory pathways.

What emerging techniques might enhance the study of REPS2 function and regulation?

Several cutting-edge methodologies offer exciting opportunities to advance our understanding of REPS2 biology:

• Proximity-Based Labeling Techniques:

  • BioID or TurboID fusion with REPS2 to identify proximal proteins in living cells

  • APEX2-REPS2 fusion for temporal mapping of the REPS2 interactome during endocytosis

  • Split-BioID systems to study context-specific interactions in different cellular compartments

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) to visualize REPS2 nanoscale organization

  • Lattice light-sheet microscopy for long-term, high-resolution imaging of REPS2 dynamics

  • Correlative light and electron microscopy (CLEM) to link REPS2 localization with ultrastructural features

• CRISPR-Based Approaches:

  • CRISPRa/CRISPRi for endogenous REPS2 expression modulation

  • CRISPR base editors to introduce specific REPS2 mutations

  • CRISPR screens to identify synthetic lethal interactions with REPS2 deficiency

• Single-Cell Technologies:

  • Single-cell RNA-seq to profile REPS2 expression heterogeneity in tumors

  • Single-cell proteomics to correlate REPS2 protein levels with other signaling components

  • Mass cytometry (CyTOF) with REPS2 antibodies to analyze rare cell populations

• Structural Biology Methods:

  • Cryo-EM to determine the structure of REPS2 in complex with binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Integrative modeling approaches combining various structural data

• In Vivo Models:

  • REPS2 conditional knockout mouse models to study tissue-specific functions

  • Patient-derived xenografts to evaluate REPS2 targeting in personalized medicine

  • In vivo CRISPR screens to identify context-dependent REPS2 functions

• Computational Approaches:

  • Machine learning algorithms to predict REPS2 interactions from multi-omics data

  • Molecular dynamics simulations to understand REPS2 conformational dynamics

  • Systems biology modeling of REPS2's role in receptor trafficking networks