VPS34 Antibody

What is VPS34 and what cellular functions does it regulate?

VPS34, also known as phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), is a class III PI3K that mediates the formation of phosphatidylinositol 3-phosphate. This protein serves as the catalytic subunit of different PI3K complex forms that play crucial roles in multiple membrane trafficking pathways. The PI3KC3-C1 complex is primarily involved in autophagosome initiation, while PI3KC3-C2 participates in autophagosome maturation and endocytosis . VPS34 promotes endoplasmic reticulum membrane curvature formation before vesicle budding and regulates degradative endocytic trafficking. Additionally, it is required for the abscission step in cytokinesis and participates in the transport of lysosomal enzyme precursors to lysosomes . Recent research has also implicated VPS34 activity in SARS-CoV-2 replication .

What applications is VPS34 antibody suitable for?

VPS34 antibody has been validated for multiple experimental applications:

The antibody has been extensively used in Western blot applications, with 59 publications citing its use, as well as in immunofluorescence (5 publications), immunoprecipitation (2 publications), and co-immunoprecipitation (1 publication) . When designing experiments, it's recommended to optimize antibody dilutions for each specific testing system as results may be sample-dependent .

What species reactivity has been confirmed for VPS34 antibody?

VPS34 antibody (12452-1-AP) has been tested and confirmed to react with human, mouse, and rat samples . Positive Western blot detection has been demonstrated in various cell lines and tissues including PC-3 cells, HeLa cells, Jurkat cells, human and mouse brain tissue, human and mouse testis tissue, and mouse lung tissue . For immunohistochemistry, positive detection has been shown in human prostate cancer tissue and human heart tissue . Additionally, some antibodies like ab233437 have shown reactivity with pig samples .

What is the recommended storage condition for VPS34 antibody?

VPS34 antibody should be stored at -20°C, where it remains stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is generally unnecessary for -20°C storage, simplifying laboratory handling processes. Some antibody preparations in 20μl sizes may contain 0.1% BSA . When working with the antibody, avoid repeated freeze-thaw cycles as this can degrade antibody quality and affect experimental results.

What is the molecular weight of VPS34 protein that should be detected by the antibody?

VPS34 protein has a calculated molecular weight of 100 kDa, corresponding to its 887 amino acid sequence . The observed molecular weight in experimental conditions also appears at approximately 100 kDa . When performing Western blot validation of new antibody lots or in new experimental systems, researchers should expect to observe a band at this molecular weight. Significant deviation from this expected size may indicate issues with antibody specificity, protein degradation, or post-translational modifications affecting migration patterns.

How can VPS34 phosphorylation status be assessed using specialized antibodies?

Researchers interested in VPS34 regulation can utilize phospho-specific antibodies to monitor its post-translational modifications. For instance, phosphorylation at Thr159 by Cdk1 can be detected using an anti-pT159-Vps34 antibody . When performing such experiments, it's advisable to include appropriate controls, such as:

• Incubating recombinant VPS34 with Cdk1/cyclin B complex to serve as a positive control

• Including roscovitine (a Cdk inhibitor) treatment to demonstrate specificity of the phosphorylation

• Performing phosphatase treatment to confirm that the signal is indeed due to phosphorylation

To further validate the specificity of Cdk1-mediated phosphorylation, immunodepletion of Cdk1 from mitotic cell lysates can be conducted, which should reduce VPS34 phosphorylation signals . These methodological approaches provide robust evidence for kinase-specific modifications of VPS34.

What experimental strategies can distinguish between different VPS34 complex forms?

VPS34 exists in two distinct complexes (PI3KC3-C1 and PI3KC3-C2) with different biological functions. To differentiate between these complexes, researchers can employ the following approaches:

• Co-immunoprecipitation with complex-specific partners: UVRAG is specific to PI3KC3-C2, while ATG14-L is specific to PI3KC3-C1

• Subcellular fractionation followed by Western blotting to analyze compartment-specific distribution of VPS34 and its complex partners

• Proximity ligation assays to visualize specific VPS34 complexes in situ

• Targeted inhibition of complex-specific components (e.g., ATG14-L or UVRAG) using siRNA/shRNA followed by functional assays to distinguish complex-specific roles

When interpreting results, researchers should consider that disruption of VPS34 complexes can be observed by decreased expression of associated proteins UVRAG and ATG14-L, as demonstrated in VPS34 knockdown experiments .

What controls should be included when using VPS34 antibodies to study autophagy?

When utilizing VPS34 antibodies to investigate autophagy, several critical controls should be incorporated:

• Positive controls: Include treatments known to induce autophagy (e.g., starvation, rapamycin) to demonstrate appropriate VPS34 localization or activity changes

• Negative controls: Include autophagy inhibitors (e.g., 3-methyladenine) or genetic knockdown of essential autophagy genes (e.g., ATG5, ATG7)

• Specificity controls: Use VPS34 knockdown or knockout cells to validate antibody specificity

• Pathway validation: Monitor downstream autophagy markers (LC3-II, p62/SQSTM1) in parallel to correlate VPS34 activity with autophagy progression

• Chemical inhibition controls: Include VPS34-specific inhibitors (e.g., VPS34-IN1, autophinib, PIK-III) to validate functional relationships

Research has shown that VPS34 inhibition by VPS34-IN1 inhibits both basal and L-asparaginase-induced autophagy in AML cells , serving as a useful experimental manipulation to establish VPS34's role in autophagy contexts.

How can researchers address the challenge of complete VPS34 inhibition versus partial knockdown?

Complete and acute inhibition of VPS34 may produce different cellular responses compared to partial or gradual knockdown, presenting an important experimental consideration. Research has demonstrated that:

• Doxycycline-inducible VPS34 shRNA can achieve >80% protein reduction after 4-7 days, but this gradual reduction often fails to suppress cell proliferation or induce apoptosis due to compensatory mechanisms

• Cells with reduced VPS34 levels can still maintain autophagy function, suggesting that complete inhibition may be necessary to observe certain phenotypes

• Attempts to generate homozygous VPS34 knockout cells using CRISPR/Cas9 have been largely unsuccessful, indicating that complete loss of VPS34 may be lethal in many cell types

To address these challenges, researchers should:

• Use chemical inhibitors (VPS34-IN1, autophinib, PIK-III) for acute and complete inhibition studies

• Generate heterozygous knockout or hypomorphic mutants for partial loss-of-function studies

• Consider inducible systems with dose-dependent protein reduction

• Employ complementation studies with wild-type or mutant VPS34 to validate specificity

What methodological approaches can distinguish VPS34-mediated effects from those of other PI3K family members?

Distinguishing VPS34 (class III PI3K) functions from other PI3K family members requires careful experimental design:

Research has shown that cells with heterozygous VPS34 deletions exhibit enhanced sensitivity to VPS34-IN1 compared to control cells, supporting the specificity of this inhibitor .

How is VPS34 antibody used to investigate cancer biology?

VPS34 antibody serves as a valuable tool in cancer research, particularly in studying autophagy regulation and vesicular trafficking in malignant cells. Research applications include:

• Expression analysis: VPS34 antibodies have been used to detect protein expression in various cancer cell lines, including PC-3 (prostate cancer), HeLa (cervical cancer), and Jurkat (T-cell leukemia), as well as in human prostate cancer tissue samples .

• Therapeutic target validation: In acute myeloid leukemia (AML) research, VPS34 antibodies help monitor protein levels during inhibitor studies. VPS34 inhibition induces apoptosis in AML cells but not in normal CD34+ hematopoietic cells, suggesting therapeutic potential .

• Signaling pathway analysis: VPS34 antibodies have been used to investigate how VPS34 inhibition affects crucial cancer signaling pathways. For example, VPS34-IN1 specifically inhibits STAT5 phosphorylation downstream of FLT3-ITD signaling in AML, providing insights into leukemia oncogenesis .

• Cell death mechanism studies: Combined with caspase activation assays and mitochondrial depolarization measurements, VPS34 antibodies help elucidate how VPS34 inhibition induces mitochondrial apoptotic cell death in cancer cells .

When designing cancer research experiments using VPS34 antibodies, researchers should include appropriate cancer and normal tissue controls and consider the specific cellular contexts relevant to their cancer model.

What are the technical considerations when using VPS34 antibody for immunohistochemistry in disease tissues?

When performing immunohistochemistry with VPS34 antibody on disease tissues, researchers should consider these technical aspects:

• Antigen retrieval method: For optimal results with VPS34 antibody in tissue sections, it's recommended to use TE buffer at pH 9.0 for antigen retrieval. Alternatively, citrate buffer at pH 6.0 can be used if TE buffer yields suboptimal results .

• Antibody dilution range: For immunohistochemistry applications, VPS34 antibody should be diluted within the range of 1:100-1:400 . Optimal dilution may vary based on tissue type and fixation method.

• Positive control tissues: Human prostate cancer tissue and human heart tissue have been validated as positive controls for VPS34 antibody in IHC applications . Including these tissues helps validate staining protocols.

• Background reduction: Non-specific background can be minimized by:

  • Using appropriate blocking sera matched to the host species of the secondary antibody

  • Including a peroxidase blocking step if using HRP-based detection systems

  • Optimizing antibody concentrations through titration experiments

• Detection systems: When studying disease tissues where VPS34 expression may be altered, sensitive detection systems (such as polymer-based or tyramide signal amplification) may be required to detect low expression levels accurately.

How can researchers interpret conflicting VPS34 antibody data in disease models?

When faced with conflicting VPS34 antibody results across different disease models or experimental systems, researchers should systematically evaluate:

• Antibody epitope variations: Different VPS34 antibodies may target distinct epitopes. For example, antibody 12452-1-AP targets the C-terminal region , while ab233437 recognizes a region within amino acids 600 to the C-terminus . Epitope accessibility may vary in different cellular contexts.

• Post-translational modifications: VPS34 undergoes phosphorylation at sites like Thr159 by Cdk1 , which might affect antibody recognition in different cellular states or disease conditions.

• Complex formation interference: VPS34 functions within distinct protein complexes (PI3KC3-C1 and PI3KC3-C2) , which might mask antibody epitopes differently in various cellular contexts.

• Experimental validation approaches:

  • Test multiple antibodies targeting different VPS34 epitopes

  • Validate with genetic knockdown/knockout controls

  • Compare results across multiple detection methods (WB, IHC, IF)

  • Perform antigen competition assays to confirm specificity

• Disease-specific contextual factors: Consider how disease-specific factors might alter VPS34 expression, localization, or complex formation. For example, in AML cells, VPS34 inhibition specifically affects STAT5 phosphorylation downstream of FLT3-ITD signaling , which might not be observed in other disease contexts.

What are common causes of weak or absent VPS34 signal in Western blotting?

When encountering weak or absent VPS34 signals in Western blotting, consider these potential issues and solutions:

VPS34 has been successfully detected in multiple cell lines and tissues including PC-3 cells, HeLa cells, Jurkat cells, and brain tissue from human, mouse, and rat . These samples can serve as positive controls when troubleshooting.

How should researchers optimize VPS34 immunoprecipitation protocols?

Optimizing VPS34 immunoprecipitation requires careful consideration of several parameters:

• Lysis buffer selection:

  • Use buffers containing non-ionic detergents (e.g., NP-40 or Triton X-100) at 0.5-1%

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying VPS34 phosphorylation states

• Antibody selection and amount:

  • VPS34 antibodies validated for IP applications have been cited in publications

  • Typically use 2-5 μg antibody per 500 μg-1 mg of total protein

  • Consider using magnetic beads for gentler handling than agarose

• Complex preservation considerations:

  • If studying VPS34 complexes, use milder lysis conditions to preserve protein-protein interactions

  • Monitor co-immunoprecipitation of known complex components (UVRAG, ATG14-L)

• Controls to include:

  • IgG control from same species as VPS34 antibody

  • Input sample (typically 5-10% of IP material)

  • For phosphorylation studies, include phosphatase-treated samples as negative controls

• Validation approaches:

  • Confirm successful IP by immunoblotting with a different VPS34 antibody targeting a distinct epitope

  • Verify complex integrity by blotting for known VPS34 interaction partners

What strategies can improve specificity in VPS34 immunofluorescence staining?

To enhance specificity and reduce background in VPS34 immunofluorescence experiments:

• Fixation optimization:

  • Test both paraformaldehyde (4%) and methanol fixation methods

  • For membrane structures where VPS34 localizes, gentle fixation may better preserve epitopes

• Permeabilization considerations:

  • Use mild detergents (0.1-0.3% Triton X-100 or 0.05% saponin)

  • Optimize permeabilization time (typically 5-15 minutes)

• Blocking efficiency:

  • Use 5-10% normal serum from secondary antibody host species

  • Consider adding 0.1-0.3% BSA to reduce non-specific binding

  • Include 0.1% Tween-20 in blocking buffer to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Use recommended dilution range (1:300-1:1200)

  • Validate in positive control cells (PC-3 cells have shown positive IF/ICC staining)

• Validation controls:

  • Include VPS34 knockdown cells as negative controls

  • Perform peptide competition assays to confirm specificity

  • Co-stain with markers of VPS34-positive structures (e.g., early endosomes, autophagosomes)

• Signal-to-noise enhancement:

  • Consider tyramide signal amplification for weak signals

  • Use confocal microscopy to improve resolution of VPS34-positive structures

  • Employ deconvolution algorithms to improve image quality

How can researchers validate specificity of VPS34 antibody in their experimental system?

Thorough validation of VPS34 antibody specificity in new experimental systems should include:

• Genetic approach validation:

  • Test antibody in VPS34 knockdown/knockout systems

  • Complete VPS34 knockout is challenging as it may be lethal , so partial knockdown or heterozygous deletion models may be more practical

  • Cells with partial VPS34 reduction show enhanced sensitivity to VPS34-IN1, which can serve as a functional validation

• Multi-antibody validation:

  • Test multiple antibodies targeting different VPS34 epitopes

  • Compare results between C-terminal targeting antibodies (like 12452-1-AP) and antibodies recognizing other regions

• Cross-species reactivity:

  • Confirm expected cross-reactivity in human, mouse, and rat samples

  • When working with other species, perform additional validation steps

• Molecular weight confirmation:

  • Verify detection at the expected molecular weight (100 kDa)

  • Use recombinant VPS34 protein as a positive control

• Functional correlation:

  • Correlate antibody detection with VPS34 functional readouts (e.g., autophagy markers, PI3P production)

  • Use specific VPS34 inhibitors (VPS34-IN1, autophinib, PIK-III) to validate functional relationships

• Phosphorylation-specific validation:

  • For phospho-specific antibodies, include phosphatase-treated controls

  • Validate with kinase treatment (e.g., Cdk1/cyclin B for Thr159 phosphorylation)

How are VPS34 antibodies being used to study the relationship between VPS34 and SARS-CoV-2 infection?

Recent research has identified VPS34 as a potential factor in SARS-CoV-2 replication . Researchers investigating this relationship can utilize VPS34 antibodies to:

• Infection-induced changes: Monitor VPS34 expression, localization, and complex formation alterations during SARS-CoV-2 infection using immunoblotting and immunofluorescence

• Viral replication mechanisms: Investigate how VPS34-dependent membrane trafficking and autophagy pathways contribute to viral replication compartment formation

• Inhibitor studies: Combine VPS34 antibodies with VPS34 inhibitors (VPS34-IN1, autophinib, PIK-III) to correlate protein inhibition with viral replication reduction

• Interaction analysis: Use co-immunoprecipitation with VPS34 antibodies to identify potential interactions between VPS34 complexes and viral proteins

• Phosphorylation dynamics: Examine whether SARS-CoV-2 infection alters VPS34 phosphorylation status, potentially through disruption of cell cycle regulators like Cdk1

When designing these experiments, researchers should include appropriate controls such as mock-infected cells, cells treated with other respiratory viruses, and VPS34 knockdown controls to establish specificity of the observed effects.

What are the considerations when using VPS34 antibody to study autophagy dynamics in complex disease models?

When investigating autophagy dynamics in complex disease models using VPS34 antibodies, researchers should consider:

• Context-dependent regulation:

  • VPS34 exists in different complexes with distinct functions in autophagy (PI3KC3-C1) versus endocytosis (PI3KC3-C2)

  • Disease states may differentially affect these complexes

  • Co-stain with markers of different VPS34 complexes (ATG14-L for PI3KC3-C1, UVRAG for PI3KC3-C2)

• Tissue-specific considerations:

  • VPS34 antibody has been validated in diverse tissues including brain, testis, lung, and prostate cancer

  • Tissue-specific autophagy regulation may require optimization of staining protocols

  • Include appropriate tissue-matched controls

• Dynamic measurement approaches:

  • Combine VPS34 antibody staining with autophagic flux assays (e.g., LC3 turnover)

  • Use time-course experiments to capture temporal dynamics

  • Consider live-cell imaging with fluorescently tagged VPS34 to complement antibody-based fixed-cell approaches

• Inhibitor utilization strategies:

  • VPS34-IN1 inhibits both basal and stress-induced autophagy (such as L-asparaginase-induced autophagy in AML)

  • Use inhibitors at different time points to dissect initiation versus maturation roles

  • Compare effects with other autophagy inhibitors (e.g., chloroquine) to distinguish VPS34-specific effects

• Disease-specific pathway interactions:

  • In AML, VPS34 inhibition affects STAT5 phosphorylation downstream of FLT3-ITD signaling

  • Investigate disease-specific signaling nodes that may interact with VPS34-mediated autophagy

How can researchers use VPS34 antibody to investigate the relationship between autophagy and cell death mechanisms?

VPS34 antibodies provide valuable tools for exploring the complex relationship between autophagy and cell death:

• Differential response analysis:

  • VPS34-IN1 induces apoptosis in AML cells but not in normal CD34+ hematopoietic cells

  • Use VPS34 antibodies to correlate protein expression/localization with differential cell death susceptibility

• Cell death mechanism determination:

  • Research shows VPS34-IN1-induced cell death is inhibited by the pan-caspase inhibitor Q-VAD-OPH but not by inhibitors of autophagy (chloroquine), necroptosis (necrostatin-1), or ferroptosis (ferrostatin-1)

  • Combine VPS34 antibodies with markers of different cell death pathways to characterize mechanism shifts

• Temporal relationship analysis:

  • Use time-course experiments with VPS34 antibodies to determine whether autophagy inhibition precedes or follows cell death marker appearance

  • Monitor VPS34 complex integrity during cell death progression

• Compartment-specific studies:

  • Employ subcellular fractionation followed by immunoblotting with VPS34 antibodies to track protein redistribution during autophagy-cell death crosstalk

  • Use immunofluorescence to visualize VPS34 translocation between compartments

• Pathway integration analysis:

  • VPS34-IN1 affects multiple cellular functions including autophagy, vesicular trafficking, and mTORC1 signaling

  • Use VPS34 antibodies alongside pathway-specific markers to determine which VPS34-dependent function most closely correlates with cell death