ATP6V1D Antibody

What is ATP6V1D and what cellular functions does it mediate?

ATP6V1D (also known as ATP6M, VATD, or V-ATPase subunit D) is a 28-35 kDa subunit of the V-type proton ATPase complex. This complex plays essential roles in:

• Lysosomal acidification through proton translocation

• Endosomal trafficking and protein sorting

• Macroautophagic/autophagic flux maintenance

• Cellular homeostasis regulation

In particular, research has shown that ATP6V1D enhances hepatocellular carcinoma stemness and progression by maintaining autophagic flux through two mechanisms: promoting lysosomal acidification and enhancing the interaction between CHMP4B and IST1 to foster ESCRT-III complex assembly, thereby facilitating autophagosome-lysosome fusion .

Which experimental applications have been validated for ATP6V1D antibodies?

ATP6V1D antibodies have been validated for multiple research applications with specific recommended protocols:

The cross-species reactivity has been confirmed for human, mouse, and rat samples .

How should researchers select appropriate controls when using ATP6V1D antibodies?

For rigorous experimental design with ATP6V1D antibodies:

• Positive tissue controls: Human brain tissue and lung cancer samples have shown consistent ATP6V1D expression

• Negative controls: Include isotype control (rabbit IgG) at equivalent concentrations

• Knockdown validation: Compare expression in wild-type vs ATP6V1D-silenced samples

• Loading controls: For Western blots, GAPDH has been validated as an appropriate normalization control

• Blocking peptide controls: Pre-incubation with immunizing peptide (ATP6V1D recombinant protein) to confirm specificity

• Cross-validation: Compare results using different antibody clones targeting distinct epitopes of ATP6V1D

Cross-validation is particularly important as ATP6V1D is part of a complex with multiple similar subunits .

What protocol modifications improve Western blot detection of ATP6V1D?

Optimizing Western blot protocols for ATP6V1D detection:

• Sample preparation: Use lysis buffers containing protease inhibitors to prevent degradation

• Protein denaturation: Heat samples at 95°C for 5 minutes in sample buffer containing 5% β-mercaptoethanol

• Gel selection: 10-12% polyacrylamide gels provide optimal resolution for 28-35 kDa proteins

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

• Membrane blocking: Block with 5% non-fat milk or 3% BSA in TBST for 1 hour at room temperature

• Antibody incubation: Primary antibody (1:1000) overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody

• Signal detection: Enhanced chemiluminescence with exposure times adjusted based on expression levels

Recent studies achieved clear detection in human brain tissue at 1:500 dilution with minimal background .

How can researchers optimize immunohistochemistry protocols for ATP6V1D localization studies?

For high-resolution ATP6V1D localization:

• Fixation: 4% paraformaldehyde for 15-30 minutes (frozen sections) or formalin-fixed paraffin embedding

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 15-20 minutes

• Permeabilization: 0.5% Triton X-100 for 10 minutes at room temperature

• Blocking: 3% BSA for 30 minutes to reduce non-specific binding

• Primary antibody: Incubate with ATP6V1D antibody (1:100) overnight at 4°C

• Secondary detection: Appropriate species-specific secondary antibody (1:1000)

• Co-staining: Combine with lysosomal markers (LAMP1) to assess subcellular localization

• Mounting: Use anti-fade mounting medium containing DAPI for nuclear visualization

This approach has successfully revealed ATP6V1D distribution in human lung cancer tissue with high specificity .

What considerations are important when designing co-localization experiments with ATP6V1D antibodies?

For meaningful co-localization studies:

• Compatible antibody pairs: Select ATP6V1D antibody and co-staining markers from different host species

• Sequential staining: For antibodies from the same species, use sequential immunostaining with blocking steps

• Fluorophore selection: Choose fluorophores with minimal spectral overlap

• Confocal microscopy settings: Optimize laser power, detector gain, and pinhole settings for each channel

• Quantification methods: Use Pearson's correlation coefficient or Manders' overlap coefficient for quantitative assessment

• Validation controls: Include single-stained samples to rule out bleed-through

• Biological controls: Compare ATP6V1D localization under different physiological or pathological conditions

Recent studies demonstrated successful co-localization of ATP6V1D with lysosomal markers using rabbit polyclonal anti-ATP6V1B2 antibody and mouse monoclonal anti-Lamp1 antibody .

How can researchers investigate ATP6V1D's role in the autophagy-lysosomal pathway?

To dissect ATP6V1D's function in autophagy:

• Genetic manipulation approaches:

  • siRNA/shRNA-mediated knockdown (transient or stable)

  • CRISPR-Cas9 knockout or knockin of mutant variants

  • Overexpression of wild-type or mutant ATP6V1D

• Autophagy flux assessment:

  • Monitor LC3-II/LC3-I conversion by Western blot

  • Track p62/SQSTM1 accumulation or degradation

  • Use tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish autophagosome formation from fusion

  • Measure long-lived protein degradation rates

• Lysosomal function evaluation:

  • LysoTracker staining to assess lysosomal acidification

  • Cathepsin activity assays to measure lysosomal enzyme function

  • Lysosomal membrane permeabilization assessment

• Autophagosome-lysosome fusion analysis:

  • Co-localization of LC3 and LAMP1

  • Electron microscopy for ultrastructural analysis

  • Live-cell imaging of fusion events

• Pharmacological interventions:

  • Bafilomycin A1 for V-ATPase inhibition (positive control)

  • Chloroquine or NH₄Cl to neutralize lysosomal pH

  • 3-methyladenine (3-MA) to inhibit autophagosome formation

Recent studies have shown that ATP6V1D not only promotes lysosomal acidification but also enhances the interaction between CHMP4B and IST1 to foster ESCRT-III complex assembly, facilitating autophagosome-lysosome fusion .

What approaches are effective for studying ATP6V1D's role in cancer stemness?

For investigating ATP6V1D in cancer stem cell biology:

• Expression correlation analysis:

  • Assess correlation between ATP6V1D and established stemness markers (PROM1, CD24, EPCAM, CD13, CD44, THY1)

  • Analyze patient-derived samples with varying degrees of stemness

• Functional stemness assays:

  • Sphere formation assays following ATP6V1D modulation

  • Colony formation efficiency

  • Limiting dilution tumor initiation assays in vivo

  • Serial transplantation experiments

• Signaling pathway investigation:

  • Examine effects on Wnt/β-catenin, Notch, and Hedgehog pathways

  • Analyze transcription factors regulating stemness (OCT4, SOX2, NANOG)

• Therapeutic implications:

  • Test low-dose bafilomycin A1 effects on cancer stemness

  • Evaluate combination approaches with conventional chemotherapeutics

Pearson correlation analysis has shown that ATP6V1D expression positively correlates with cancer stem cell markers including PROM1, CD24, EPCAM, CD13, CD44, and THY1, suggesting its potential role in maintaining cancer stem cell properties .

What methodologies enable investigation of protein-protein interactions involving ATP6V1D?

To characterize ATP6V1D's interactome:

• Co-immunoprecipitation (co-IP):

  • Use anti-Flag antibodies for tagged ATP6V1D constructs

  • Apply stringent washing conditions to reduce false positives

  • Verify interactions by reciprocal co-IP

• Proximity-dependent labeling:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2-based proximity labeling for temporal dynamics

• FRET/FLIM analysis:

  • Fluorescently tag ATP6V1D and potential interaction partners

  • Measure energy transfer as indicator of protein proximity

• Mass spectrometry-based approaches:

  • Label-free quantitative proteomics

  • SILAC labeling for quantitative comparison

  • Crosslinking mass spectrometry for structural information

• Structural biology methods:

  • X-ray crystallography of ATP6V1D complexes

  • Cryo-EM analysis of V-ATPase assemblies

  • Hydrogen-deuterium exchange mass spectrometry

Recent research demonstrated successful co-IP of ATP6V1D with CHMP4B and IST1, revealing its role in enhancing ESCRT-III complex assembly for autophagosome-lysosome fusion .

How should researchers address common technical challenges with ATP6V1D antibodies?

These troubleshooting approaches are based on validated protocols established with commercial ATP6V1D antibodies .

How can researchers interpret conflicting data regarding ATP6V1D function?

When encountering contradictory results:

• Biological context consideration:

  • Cell/tissue-specific functions may vary dramatically

  • Disease stage may influence ATP6V1D behavior (particularly in cancer progression)

  • Acute vs. chronic modulation may have opposite effects

• Technical assessment:

  • Antibody specificity confirmation through multiple methods

  • Knockdown/knockout efficiency verification

  • Cross-validation with multiple experimental approaches

• Functional redundancy analysis:

  • Assess compensatory upregulation of other V-ATPase subunits

  • Evaluate potential adaptive mechanisms following ATP6V1D modulation

• Data integration strategies:

  • Weigh evidence based on methodological rigor

  • Consider time-dependent effects in experimental design

  • Integrate findings across multiple model systems

• Mechanistic reconciliation:

  • ATP6V1D may have V-ATPase-dependent and independent functions

  • Different protein interactions may predominate in different contexts

  • Post-translational modifications may alter protein function

Research has shown that ATP6V1D has dual mechanisms - promoting lysosomal acidification and enhancing autophagosome-lysosome fusion through ESCRT-III complex assembly - which may explain seemingly conflicting observations in different experimental systems .

What are promising therapeutic approaches targeting ATP6V1D or V-ATPase in disease?

Emerging therapeutic strategies include:

• V-ATPase inhibition:

  • Low-dose bafilomycin A1 showed promise for HCC treatment

  • Concanamycin A derivatives with improved pharmacokinetics

  • Subunit-specific inhibitors to reduce systemic toxicity

• Genetic modulation approaches:

  • siRNA/shRNA targeting ATP6V1D for transient knockdown

  • Antisense oligonucleotides for selective inhibition

  • PROTAC-based degradation of ATP6V1D protein

• Combination therapies:

  • V-ATPase inhibitors with autophagy modulators

  • Synergistic effects with conventional chemotherapeutics

  • Targeting both ATP6V1D and its interaction partners (CHMP4B/IST1)

• Biomarker development:

  • ATP6V1D expression as prognostic indicator in HCC

  • Correlation with treatment response prediction

  • Patient stratification for personalized therapy

Recent research demonstrated that low-dose bafilomycin A1 targeting the V-ATPase complex shows promise as a potential therapeutic strategy for hepatocellular carcinoma, highlighting ATP6V1D as an emerging therapeutic target .

How can ATP6V1D antibodies advance understanding of lysosome-related disorders?

ATP6V1D antibodies enable investigation of:

• Neurodevelopmental disorders:

  • ATP6V1 family proteins (ATP6V1C1/ATP6V1B2) are implicated in neurodevelopmental phenotypes resembling DOORS syndrome

  • ATP6V1D may have parallel functions in neuronal development and function

• Lysosomal storage diseases:

  • Detection of altered V-ATPase subunit expression or localization

  • Assessment of compensatory mechanisms in primary lysosomal disorders

  • Evaluation of lysosomal morphology and distribution changes

• Autophagy-related pathologies:

  • Investigation of defective autophagic flux in neurodegenerative diseases

  • Analysis of ATP6V1D expression in models of impaired autophagy

  • Therapeutic modulation of V-ATPase activity to restore autophagic function

• Cilium biogenesis and ciliopathies:

  • Assessment of ATP6V1D's role in cilium formation

  • Investigation of overlap between V-ATPase function and ciliopathies

Recent studies have shown that mutations in ATP6V1 family proteins affect lysosomal morphology, localization, and function, resulting in defective autophagic flux and accumulation of lysosomal substrates. This work also showed that upregulated V-ATPase function affects cilium biogenesis, documenting pleiotropy .

What next-generation experimental approaches will advance ATP6V1D research?

Cutting-edge methodologies for ATP6V1D investigation:

• Single-cell analysis:

  • Single-cell RNA-seq to map ATP6V1D expression heterogeneity

  • Single-cell proteomics for protein-level analysis

  • Spatial transcriptomics to assess tissue-specific expression patterns

• Advanced imaging technologies:

  • Super-resolution microscopy for nanoscale localization

  • Live-cell pH sensors to monitor V-ATPase activity in real-time

  • Correlative light and electron microscopy for structure-function studies

• Genome editing innovations:

  • Base editing for introducing specific ATP6V1D mutations

  • Prime editing for precise genetic modifications

  • Inducible/conditional knockout systems for temporal control

• Computational approaches:

  • AI-driven protein structure prediction for ATP6V1D complexes

  • Systems biology modeling of V-ATPase in cellular networks

  • Multi-omics data integration for comprehensive pathway analysis

• Organoid and tissue-specific models:

  • Patient-derived organoids to study ATP6V1D in disease contexts

  • Tissue-specific conditional knockout models

  • Humanized mouse models for translational research

These advanced approaches will enable researchers to better understand ATP6V1D's complex roles in cellular physiology and pathology, potentially leading to novel therapeutic interventions for diseases involving V-ATPase dysfunction.