VPS4B Antibody, HRP conjugated

What is VPS4B and what cellular processes does it regulate?

VPS4B is an AAA-ATPase that functions as a critical component of the endosomal sorting complex required for transport (ESCRT) machinery. It forms heteromeric complexes with its paralog VPS4A to facilitate membrane scission events during multivesicular body (MVB) formation . VPS4B plays crucial roles in:

• Intracellular protein trafficking and receptor sorting in the endocytic pathway

• Formation of multivesicular bodies

• Cellular membrane remodeling processes

• Viral budding, particularly for retroviruses like HIV-1 that hijack the VPS pathway

• Nuclear envelope stress response and repair mechanisms

The protein interacts with CHMP1 and other ESCRT-III components to regulate multivesicular body formation, which is essential for maintaining cellular homeostasis. Its dysregulation has been implicated in various pathological conditions, including cancer progression.

What detection methods are compatible with VPS4B Antibody, HRP conjugated?

VPS4B Antibody (A-11), when conjugated to HRP, demonstrates versatility across multiple detection platforms :

The antibody specifically detects VPS4B protein from human, mouse, and rat origins. For optimal results, researchers should perform preliminary titration experiments to determine ideal antibody concentrations for their specific sample type and experimental conditions .

How can I validate the specificity of VPS4B antibodies in my experimental system?

Validating antibody specificity is critical for reliable experimental outcomes. For VPS4B antibodies, consider these methodological approaches:

• Positive and negative tissue controls: Test antibodies on tissues with known high expression (e.g., appendix) and low expression (e.g., muscle) of VPS4B .

• Genetic knockout validation: Confirm lack of VPS4B staining in cells where VPS4B has been knocked out using CRISPR/Cas9 or other gene editing methods. Research has confirmed the specificity of anti-VPS4B antibodies using HCT116 human CRC lines with inactivated VPS4B alleles .

• Comparative analysis with other antibodies: Compare staining patterns with alternative anti-VPS4B antibodies from different sources or clones.

• Immunoprecipitation followed by mass spectrometry: Verify that the immunoprecipitated protein is indeed VPS4B.

• siRNA knockdown controls: Perform parallel experiments with VPS4B-knockdown samples to demonstrate reduced signal intensity .

How can VPS4B antibodies be used to investigate synthetic lethality between VPS4A and VPS4B in cancer research?

Research has revealed a synthetic lethal interaction between VPS4A and VPS4B, presenting a potential therapeutic avenue for cancers with VPS4B deficiency . When incorporating HRP-conjugated VPS4B antibodies in such studies:

• Quantitative cancer expression profiling: Use immunohistochemistry with VPS4B antibodies to categorize tumors based on VPS4B expression levels. Studies have shown that in colorectal cancer (CRC), VPS4B protein levels are often reduced compared to normal colon tissue, with 39% of cases showing prominent decreases and 4% showing complete loss of VPS4B protein .

• Dual paralog detection assays: Simultaneously assess VPS4A and VPS4B expression levels using specific antibodies against each paralog to identify tumors that might be vulnerable to synthetic lethality-based therapeutic approaches.

• Validation in cell line models: Use VPS4B antibodies to confirm protein depletion after siRNA treatment in various cancer cell lines to establish dependency relationships. Research has demonstrated that cancer cells with low VPS4B expression become sensitized to VPS4A depletion .

• In vivo tumor response monitoring: Employ VPS4B antibodies in xenograft models to track protein expression changes during treatment with VPS4A inhibitors.

The synthetic lethal relationship between these paralogs provides a potential precision medicine approach for treating VPS4B-deficient cancers, particularly those with chromosome 18q loss containing the VPS4B locus .

What role does VPS4B play in nuclear envelope stress response, and how can VPS4B antibodies help elucidate this mechanism?

VPS4B has been identified as a key regulator in the nuclear envelope (NE) stress response . HRP-conjugated VPS4B antibodies can facilitate several advanced research approaches:

• Dynamic localization studies: Track VPS4B relocalization during NE stress events. Under normal conditions, VPS4B is distributed throughout the cell, but under NE stress (such as cellular compression), VPS4B accumulates at the nuclear envelope, colocalizing with inner nuclear membrane proteins like Lap2β .

• Stress response timing analysis: Investigate the temporal dynamics of VPS4B involvement in NE repair. Research shows that approximately 27% of cells exhibit nuclear localization of VPS4B immediately after compression, with about 40% showing NE accumulation 30 minutes post-compression .

• Functional analysis in VPS4B-deficient systems: Use VPS4B antibodies to confirm knockdown efficiency when investigating how VPS4B deficiency affects cellular response to NE stress. Studies have shown that VPS4B depletion increases the proportion of cells with γH2A.X accumulation (a DNA damage marker) from 1% to 4% under NE stress conditions .

• ESCRT-III interaction studies: Examine the relationship between VPS4B and ESCRT-III components like CHMP7 during NE repair processes, as VPS4B regulates ESCRT-III dynamics .

How do VPS4B expression levels correlate with cancer progression and therapeutic vulnerabilities?

VPS4B expression analysis using antibody-based techniques has revealed significant correlations with cancer progression and therapeutic opportunities :

• Copy number alterations: Research has shown that VPS4B copy number loss, often occurring with chromosome 18q deletion, creates a vulnerability that can be exploited by targeting VPS4A. Approximately 63.9% (106/166) of VPS4A-dependent cancer cell lines demonstrated at least partial genomic loss of VPS4B .

• Cancer-type specific dependencies: VPS4B expression varies across cancer types:

  • Over 40% of pancreatic cancers and pediatric rhabdomyosarcomas (RMS) demonstrate robust dependence on VPS4A when VPS4B is deficient

  • Substantial fractions of bladder, bile duct, lung, ovarian, colon, and esophageal cancers also show this dependency pattern

  • For VPS4B dependency (when VPS4A is deficient), over 25% of ovarian, breast, pancreatic, liver, gastric, and bile duct cancer cell lines show strong dependence

• Protein-level quantification: Immunohistochemical analysis of CRC patient samples revealed that:

  • 57% of cases showed slightly decreased VPS4B staining (3+ → 2+)

  • 39% showed prominent decrease (3+ → 1+)

  • 4% showed complete loss of VPS4B protein (3+ → 0)

These findings highlight the potential of using VPS4B antibodies to stratify tumors for potential synthetic lethal therapeutic approaches.

What are the optimal protocols for using VPS4B Antibody, HRP conjugated in Western blotting applications?

For optimal Western blotting results with HRP-conjugated VPS4B antibodies, follow these methodological recommendations:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Determine protein concentration using Bradford or BCA assay

  • Load 20-50 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute HRP-conjugated VPS4B antibody 1:500-1:1000 in blocking solution

  • Incubate membrane with antibody solution overnight at 4°C or 2 hours at room temperature

• Detection:

  • Wash membrane 3-5 times with TBST, 5 minutes each

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or capture signal using a digital imaging system

• Expected results:

  • VPS4B protein appears as a band at approximately 49 kDa

  • Signal intensity correlates with VPS4B expression levels in different cell types or experimental conditions

For validation, consider running VPS4B-knockout or siRNA-mediated knockdown samples as negative controls .

How can I optimize immunohistochemistry protocols for VPS4B detection in different tissue types?

Optimizing immunohistochemistry with HRP-conjugated VPS4B antibodies requires consideration of tissue-specific factors:

• Tissue preparation and fixation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section tissues at 4-5 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Microwave or pressure cooker treatment for 15-20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal serum for 1 hour

  • Dilute HRP-conjugated VPS4B antibody 1:50-1:200 in antibody diluent

  • Incubate sections with antibody solution overnight at 4°C

• Detection and visualization:

  • Develop signal using DAB substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount sections

• Scoring system:

  • Implement a semi-quantitative scoring method based on staining intensity:

    • 0: No staining

    • 1+: Weak staining

    • 2+: Moderate staining

    • 3+: Strong staining

For validation, include tissues with known high expression (appendix) and low expression (muscle) of VPS4B as controls .

What troubleshooting approaches should I consider when experiencing weak or non-specific signals with VPS4B antibodies?

When encountering signal issues with HRP-conjugated VPS4B antibodies, consider these methodological troubleshooting steps:

• For weak signals:

  • Increase antibody concentration (reduce dilution)

  • Extend antibody incubation time

  • Optimize antigen retrieval conditions

  • Increase protein loading for Western blots

  • Use a more sensitive detection system

  • Check sample handling to prevent protein degradation

• For non-specific signals:

  • Increase blocking time or concentration

  • Reduce antibody concentration

  • Add 0.1-0.5% Triton X-100 to reduce background

  • Perform additional washing steps

  • Pre-adsorb antibody with non-specific proteins

  • Confirm antibody specificity using knockout controls

• For inconsistent results between experiments:

  • Standardize protein extraction methods

  • Prepare fresh buffers for each experiment

  • Maintain consistent incubation times and temperatures

  • Include proper positive and negative controls

  • Consider lot-to-lot variations of antibodies

• For discrepancies between detection methods:

  • Different epitopes may be accessible in different applications

  • Native protein folding may affect antibody binding in certain techniques

  • Fixation procedures may alter epitope recognition

Remember that antibody validation should be performed for each new application, cell line, or tissue type to ensure reliable results .

How can I quantitatively analyze VPS4B expression levels in relation to ESCRT pathway dysfunction?

To quantitatively assess VPS4B expression and its relationship to ESCRT pathway function:

• Relative protein quantification:

  • Normalize VPS4B Western blot signals to loading controls (e.g., GAPDH, β-actin)

  • Use densitometry software to quantify signal intensity

  • Compare relative VPS4B levels across different experimental conditions or samples

• Multiplex analysis:

  • Perform co-immunostaining with VPS4B and other ESCRT components

  • Quantify colocalization coefficients

  • Measure correlation between VPS4B levels and other ESCRT proteins

• Functional correlation analysis:

  • Develop a standardized scoring system for ESCRT dysfunction phenotypes

  • Correlate VPS4B expression levels with:

    • Multivesicular body formation efficiency

    • Endosomal sorting defects

    • Viral budding capacity

    • Nuclear envelope repair kinetics

• Synthetic lethality quantification:

  • Create dose-response curves for VPS4A inhibition at varying VPS4B expression levels

  • Calculate IC50 values to determine the relationship between VPS4B expression and VPS4A dependency

  • Use computational modeling to predict therapeutic windows based on VPS4B expression

What approaches should I use to analyze contradictory results between VPS4B protein levels and functional outcomes?

When facing contradictory results between VPS4B protein levels and functional outcomes, consider these analytical approaches:

• Assess post-translational modifications:

  • Investigate phosphorylation status of VPS4B, which may affect its activity independent of expression level

  • Examine ubiquitination patterns that might influence protein turnover

  • Check for other modifications that could impact function without altering total protein levels

• Evaluate protein-protein interactions:

  • Analyze VPS4B's interaction with VPS4A and ESCRT-III components

  • Assess the formation of functional heteromeric complexes

  • Consider that complex formation, rather than absolute protein levels, may better correlate with function

• Measure enzymatic activity:

  • Implement the VPS4B Activity Assay, which directly detects ADP produced by VPS4B to measure enzymatic activity

  • Compare protein levels to actual enzymatic function

  • Create activity-to-expression ratios to normalize functional data

• Investigate cellular compartmentalization:

  • Assess VPS4B localization under different conditions

  • Consider that proper localization (e.g., nuclear envelope accumulation during stress) may be more important than total protein levels

  • Perform subcellular fractionation to determine the active pool of VPS4B

• Implement genetic rescue experiments:

  • Reintroduce wild-type or mutant VPS4B into depleted systems

  • Determine which protein domains are essential for specific functions

  • Assess whether contradictory results can be explained by functional redundancy with VPS4A

How should I design experiments to investigate the synthetic lethal relationship between VPS4A and VPS4B in different cancer contexts?

To rigorously investigate the synthetic lethal relationship between VPS4A and VPS4B across cancer contexts, implement these experimental design strategies:

• Genetic manipulation approaches:

  • Use CRISPR/Cas9 to create VPS4B knockout cell lines from different cancer types

  • Implement inducible shRNA or siRNA systems for controlled VPS4A depletion in VPS4B-deficient cells

  • Create isogenic cell line pairs differing only in VPS4B status

• Expression correlation analysis:

  • Quantify both mRNA and protein levels of VPS4A and VPS4B

  • Note that these paralogs undergo independent regulation

  • Remember that protein levels may not directly correlate with mRNA expression

• Synthetic lethality validation:

  • Establish dose-dependent relationships between VPS4B levels and VPS4A dependency

  • Measure cell viability, clonogenic growth, and apoptosis markers

  • Confirm synthetic lethality across multiple cell lines with diverse genetic backgrounds

• Mechanistic studies:

  • Investigate cellular consequences of dual VPS4A/B depletion:

    • ESCRT-III filament accumulation

    • Cytokinesis defects

    • Nuclear membrane abnormalities

    • Micronucleation

    • G2/M cell cycle arrest

    • Apoptosis

• In vivo validation:

  • Establish xenograft models using VPS4B-deficient cancer cells

  • Implement controllable VPS4A inhibition in established tumors

  • Monitor tumor regression and analyze mechanisms using immunohistochemistry with VPS4B antibodies

• Drug development considerations:

  • Screen for small molecule inhibitors of VPS4A

  • Test compounds in VPS4B-proficient and VPS4B-deficient contexts

  • Establish therapeutic windows based on VPS4B expression levels

This systematic approach enables robust characterization of the synthetic lethal relationship, potentially leading to novel precision medicine strategies for cancers with VPS4B deficiency .

What emerging applications of VPS4B antibodies might advance our understanding of nuclear envelope dynamics?

The discovery of VPS4B's role in nuclear envelope (NE) stress response opens several promising research avenues where VPS4B antibodies could be instrumental:

• Live-cell imaging of NE repair processes:

  • Develop non-HRP conjugated fluorescent VPS4B antibody fragments for real-time monitoring

  • Track VPS4B recruitment kinetics during NE rupture and repair

  • Correlate VPS4B dynamics with other repair factors

• Nuclear-cytoplasmic compartmentalization studies:

  • Investigate how VPS4B participates in maintaining nuclear integrity

  • Explore the relationship between VPS4B and nuclear pore complex components

  • Examine how nuclear VPS4B localization changes in response to various stressors

• Mechanistic studies of ESCRT-III regulation:

  • Use VPS4B antibodies to examine how VPS4B orchestrates ESCRT-III filament assembly and disassembly at the NE

  • Investigate the spatial and temporal regulation of CHMP7 by VPS4B during NE stress response

  • Map the interactome of nuclear VPS4B during different phases of NE repair

• Cancer cell migration and invasion models:

  • Explore how NE stress during migration through confined spaces affects VPS4B localization

  • Investigate whether VPS4B dysfunction contributes to genomic instability in cancer

  • Determine if NE repair efficiency correlates with metastatic potential

• Therapeutic targeting opportunities:

  • Identify cancer types with aberrant NE dynamics that might be vulnerable to VPS4B modulation

  • Develop strategies to exploit NE repair dependencies in specific cancer contexts

  • Explore combination approaches targeting both membrane remodeling and DNA repair pathways

How might VPS4B antibodies contribute to developing novel cancer therapeutics targeting the ESCRT pathway?

VPS4B antibodies can play critical roles in developing ESCRT-targeted cancer therapeutics:

• Patient stratification biomarkers:

  • Use VPS4B immunohistochemistry to identify tumors with reduced VPS4B expression

  • Develop standardized scoring systems to categorize tumors based on VPS4B levels

  • Create companion diagnostic tests for VPS4A-targeting therapies

• Drug discovery support:

  • Utilize antibodies in high-throughput screening assays to identify compounds that modulate VPS4B activity

  • Employ VPS4B antibodies in target engagement studies to confirm binding of candidate molecules

  • Develop activity-based protein profiling using modified VPS4B antibodies

• Mechanism-of-action studies:

  • Investigate how potential therapeutics affect VPS4B localization and function

  • Examine downstream effects on ESCRT-III dynamics and membrane remodeling

  • Monitor compensatory pathways that emerge during VPS4B/VPS4A targeting

• Therapeutic response monitoring:

  • Track changes in VPS4B expression during treatment

  • Assess whether VPS4B levels predict response to ESCRT-targeting therapies

  • Develop circulating biomarkers based on VPS4B to monitor treatment efficacy

• Combination therapy development:

  • Identify synthetic lethal interactions beyond VPS4A that could be exploited in VPS4B-deficient tumors

  • Use modifier screens similar to those that identified genes promoting or suppressing VPS4A dependency

  • Develop rational combinations targeting parallel cellular processes

The unique synthetic lethal relationship between VPS4A and VPS4B presents a promising therapeutic opportunity, particularly for the significant percentage of tumors with chromosome 18q or 16q loss .