VPS34 Antibody

Basic Research Questions

• What is VPS34 and what are its main functions in cellular processes?

  VPS34, also known as PIK3C3 (Phosphatidylinositol 3-kinase catalytic subunit type 3), is the catalytic subunit of the PI3K complex that mediates formation of phosphatidylinositol 3-phosphate. Different complex forms of VPS34 play crucial roles in multiple membrane trafficking pathways . The PI3KC3-C1 complex is involved in autophagosome initiation, while PI3KC3-C2 participates in autophagosome maturation and endocytosis .

  VPS34 promotes endoplasmic reticulum membrane curvature formation prior to vesicle budding and regulates degradative endocytic trafficking . It's also required for the abscission step in cytokinesis and is involved in the transport of lysosomal enzyme precursors to lysosomes and the transport from early to late endosomes . Recent research has also identified VPS34 as playing a significant role in metabolic regulation, as heterozygous Vps34 kinase-dead mice display enhanced insulin sensitivity and glucose tolerance .

• What applications are VPS34 antibodies commonly used for in research?

  VPS34 antibodies are utilized across multiple research applications, with varying optimal dilutions:

  Application	Dilution	Published Studies
  Western Blot (WB)	1:500-1:1000	59+ publications
  Immunohistochemistry (IHC)	1:100-1:400	Multiple studies
  Immunofluorescence (IF)/ICC	1:300-1:1200	5+ publications
  Immunoprecipitation (IP)	Variable	2+ publications
  Co-Immunoprecipitation (CoIP)	Variable	1+ publications
  ELISA	Variable	Multiple studies

  These antibodies have shown reactivity with human, mouse, and rat samples . It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as the optimal dilution can be sample-dependent .

• What are the key characteristics of commercially available VPS34 antibodies?

  Available VPS34 antibodies typically target the C-terminal region of the protein. Key characteristics include:

  • Reactivity: Tested with human, mouse, and rat samples

  • Host/Isotype: Commonly rabbit IgG

  • Class: Both polyclonal and multiclonal options available

  • Molecular Weight: Recognizes a protein of approximately 100 kDa (calculated molecular weight: 887 aa, 100 kDa)

  • Storage: Typically stable for one year after shipment when stored at -20°C

  • Format: Usually supplied in liquid form, in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  Multiclonal antibodies offer the sensitivity of polyclonal antibodies by recognizing multiple epitopes, along with the consistency of a recombinant antibody .

• What tissue and cell types commonly express VPS34 that can be detected with antibodies?

  VPS34 antibodies have been successfully used to detect the protein in multiple tissue and cell types:

  Sample Type	Validated Detection
  Cells	PC-3 cells, HeLa cells, Jurkat cells
  Human Tissues	Testis tissue, brain tissue, prostate cancer tissue, heart tissue
  Mouse Tissues	Brain tissue, lung tissue, testis tissue
  Rat Tissues	Brain tissue

  Western blot has confirmed VPS34 expression in all these tissues and cell lines . For immunohistochemistry, detection works best with antigen retrieval using TE buffer pH 9.0, although citrate buffer pH 6.0 can be used as an alternative .

Advanced Research Questions

• How can VPS34 activity be measured in experimental settings?

  VPS34 activity can be measured through lipid kinase assays using phosphatidylinositol (PI) as a substrate. A detailed protocol involves:

  1. Cell or tissue lysis in buffer containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.4, 10% Glycerol, 1 mM CaCl₂, 1 mM MgCl₂, and protease/phosphatase inhibitors

  2. Immunoprecipitation of VPS34 from 1 mg of total protein using protein A Sepharose and specific VPS34 antibodies

  3. Resuspension of beads in kinase buffer (20 mM Tris, pH 7; 67 mM NaCl; 10 mM MnCl₂; 0.02% (w/v) NP-40)

  4. Kinase assay using 0.1 μCi/μl radioactive labeled γ-ATP (³²P) per reaction for 15 min at 30°C

  5. Extraction of lipids using Chloroform-Methanol extraction protocol

  6. Separation of extracted lipids using Silica 60 thin layer chromatography (TLC)

  7. Quantification of PI3P spots using imaging systems like Typhoon Imaging System

  Additionally, monitoring SGK3 phosphorylation and activity can be employed as a biomarker of Vps34 activity, similar to how Akt is used to probe cellular class I PI3K activity .

• What are the considerations for using VPS34 inhibitors in research alongside antibodies?

  When using VPS34 inhibitors like VPS34-IN1 alongside antibodies in research, consider:

  1. Selectivity: Ensure the inhibitor is selective for VPS34 over other kinases to avoid off-target effects

  2. Dosage and timing: Determine appropriate dosage and treatment duration through pilot experiments

  3. Validation of inhibition: Use VPS34 activity assays (as described above) to confirm successful inhibition

  4. Downstream effects: Monitor effects on autophagy markers, endosomal trafficking, and PI3P levels

  5. Physiological impact: Be aware that VPS34 inhibition can alter cellular energy metabolism and activate the AMPK pathway in liver and muscle

  6. Control conditions: Include proper vehicle controls for comparison

  VPS34-IN1 has been documented as a useful probe to delineate physiological roles of Vps34 . Researchers should note that VPS34 inhibition can lead to complex cellular responses, including a mild dampening of autophagy in the liver, limited substrate availability for mitochondrial respiration, reduced gluconeogenesis, and a metabolic switch from oxidative phosphorylation towards glycolysis in muscle tissue .

• How can researchers detect VPS34 ubiquitination and what methods are available to study this modification?

  VPS34 undergoes K29/K48 branched ubiquitination, which can be studied using several approaches:

  1. Ubiquitination analysis using specific antibodies: Use K48 chain-specific antibodies to detect this linkage type on immunoprecipitated VPS34

  2. Deubiquitination assays: Employ deubiquitinases like TRABID that can remove K29- and K48-linked ubiquitin chains from VPS34 to study the dynamics of ubiquitination

  3. In vitro ubiquitination: Use purified components including E3 ligases like UBE3C that generate K29/K48 heterotypic ubiquitin chains to study the process in a controlled setting

  4. Mass spectrometry analysis of ubiquitin chain topology:

     • Use Lb pro* protease treatment which cleaves ubiquitin after Arg74

     • This disassembles polyubiquitin chains into GG-modified monoubiquitin

     • Ubiquitin at branched points generates multiple GG-modified species

     • Detect these species using intact mass MS analysis

     • Quantify by peak integration to determine the percentage of branched ubiquitin

  This advanced methodology has been validated by analyzing free polyubiquitin chains assembled by UBE3C in vitro, where double GG-modified ubiquitin species represented 12.5% of the total ubiquitin .

• What are the key considerations for optimizing immunofluorescence protocols using VPS34 antibodies?

  For optimal immunofluorescence results with VPS34 antibodies, consider:

  1. Sample preparation: Culture cells on glass coverslips and process using standard immunocytochemistry protocols

  2. Dilution optimization: Test a range of dilutions from 1:300-1:1200 to determine optimal signal-to-noise ratio for your specific cell type

  3. Fixation method: Compare paraformaldehyde (PFA) fixation with alternative methods to determine which best preserves VPS34 antigenicity while maintaining cellular structures

  4. Permeabilization: Optimize detergent type and concentration for accessing intracellular VPS34 while preserving membranous structures where VPS34 localizes

  5. Blocking conditions: Test different blocking solutions to minimize background signal

  6. Co-staining strategies: Consider co-staining with markers of specific cellular compartments (endosomes, autophagosomes) to analyze VPS34 localization

  7. Positive controls: Include cells with known VPS34 expression patterns such as PC-3 cells, which have been validated for IF/ICC with VPS34 antibodies

  8. Negative controls: Include samples without primary antibody and, if possible, VPS34-depleted cells using siRNA or CRISPR knockout

  These optimizations are crucial for achieving reliable and interpretable results when studying VPS34 localization and dynamics within cells.

• How can researchers distinguish between the different VPS34 complexes (PI3KC3-C1 vs PI3KC3-C2) in experimental settings?

  Distinguishing between PI3KC3-C1 and PI3KC3-C2 complexes requires sophisticated experimental approaches:

  1. Co-immunoprecipitation with complex-specific partners:

     • PI3KC3-C1 contains VPS34, VPS15, Beclin-1, and ATG14

     • PI3KC3-C2 contains VPS34, VPS15, Beclin-1, and UVRAG

     • Immunoprecipitate with antibodies against ATG14 or UVRAG to pull down specific complexes

  2. Functional assays based on known complex roles:

     • PI3KC3-C1: Measure autophagosome initiation

     • PI3KC3-C2: Assess autophagosome maturation and endocytosis

  3. Subcellular localization studies:

     • Use immunofluorescence with antibodies against complex-specific components

     • PI3KC3-C1 primarily localizes to ER and early autophagic structures

     • PI3KC3-C2 is more associated with endosomes and mature autophagic structures

  4. Specific inhibitor response:

     • Some inhibitors might have differential effects on each complex

     • Monitor complex-specific endpoints after inhibitor treatment

  5. Genetic manipulation approaches:

     • Selectively deplete complex-specific components (ATG14 or UVRAG)

     • Assess effects on VPS34 localization and function

  These methodologies help researchers investigate the distinct roles of each complex in membrane trafficking, autophagy, and other cellular processes .

• What methodological approaches can researchers use to study the role of VPS34 in insulin sensitivity and metabolic regulation?

  Research into VPS34's role in metabolism can employ several methodological approaches:

  1. Genetic models: Use heterozygous Vps34 kinase-dead mice (Vps34^D761A/+) which display enhanced insulin sensitivity and glucose tolerance

  2. Pharmacological inhibition: Administer selective Vps34 inhibitors to wild-type mice to mimic the genetic model phenotypes

  3. Metabolic assays:

     • Glucose tolerance tests

     • Insulin tolerance tests

     • Measurement of hepatic glucose production

  4. Mechanistic studies:

     • Assess cellular energy metabolism parameters

     • Monitor AMPK pathway activation in liver and muscle

     • Examine autophagy markers to detect mild dampening of the process

     • Measure mitochondrial respiration and substrate availability

     • Analyze muscle tissue for metabolic switching from oxidative phosphorylation to glycolysis

  5. Signaling pathway analysis:

     • Western blot analysis of insulin signaling components (Akt phosphorylation on S473 and T308)

     • Assessment of downstream targets (GSK3α/β on S21 and S29, AS160 on T642, PRAS40 on T246)

     • Evaluation of mTORC1 signaling (S6K phosphorylation on T389, S6 on S240 and S244)

  It's important to note that the insulin sensitization observed in Vps34^D761A/+ mice operates independently of insulin-mediated Akt/mTORC1 signaling , suggesting that researchers should explore alternative mechanisms such as AMPK activation when studying VPS34's metabolic functions.

• What are the best practices for troubleshooting non-specific binding or background issues when using VPS34 antibodies?

  When encountering specificity or background issues with VPS34 antibodies, consider these methodological approaches:

  1. Antibody validation:

     • Verify antibody specificity using VPS34 knockout or knockdown samples

     • Test multiple antibodies targeting different VPS34 epitopes

     • Consider multiclonal antibodies which offer both sensitivity and consistency

  2. Protocol optimization:

     • Western blot: Adjust blocking conditions (5% milk vs. BSA), incubation times, washing stringency

     • IHC: Test different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

     • IF/ICC: Optimize fixation, permeabilization, and blocking procedures

  3. Sample preparation refinement:

     • Ensure proper tissue or cell lysis conditions

     • For IP applications, modify lysis buffer components (1% Triton X-100, 150 mM NaCl, etc.)

     • Consider fresh vs. frozen samples for optimal protein preservation

  4. Application-specific considerations:

     • WB: Test dilutions between 1:500-1:1000

     • IHC: Try dilutions in the 1:100-1:400 range

     • IF/ICC: Explore dilutions from 1:300-1:1200

  5. Controls implementation:

     • Include peptide competition assays to confirm epitope specificity

     • Run no-primary-antibody controls for background assessment

     • Include isotype controls to identify non-specific binding

  These troubleshooting approaches should be systematically applied and documented to identify the optimal conditions for specific VPS34 detection in your experimental system.

Critical Considerations for Method Implementation

• How should researchers design experiments to study the relationship between VPS34 and autophagy regulation?

  When investigating VPS34's role in autophagy, consider these methodological approaches:

  1. Genetic manipulation strategies:

     • Use Vps34 kinase-dead models (D761A mutation) to study partial loss of function

     • Compare heterozygous versus homozygous models to assess dose-dependent effects

     • Employ tissue-specific knockout models to avoid systemic effects

  2. Pharmacological approaches:

     • Utilize selective VPS34 inhibitors like VPS34-IN1

     • Compare acute versus chronic inhibition effects

     • Establish dose-response relationships

  3. Autophagy assessment methods:

     • Monitor LC3-I to LC3-II conversion via Western blot

     • Track autophagic flux using chloroquine or bafilomycin A1

     • Assess p62/SQSTM1 levels as autophagy substrate

     • Implement fluorescent reporters (GFP-LC3, mRFP-GFP-LC3)

     • Quantify autophagosomes and autolysosomes via transmission electron microscopy

  4. PI3P production measurement:

     • Use lipid kinase assays with PI as substrate

     • Implement PI3P-binding domain reporters (FYVE, PX domains)

     • Quantify PI3P via mass spectrometry

  5. Complex-specific analysis:

     • Distinguish PI3KC3-C1 (autophagosome initiation) from PI3KC3-C2 (maturation) functions

     • Manipulate complex-specific components (ATG14 for C1, UVRAG for C2)

  Remember that VPS34 inhibition has multifaceted effects beyond autophagy, including altered cellular energy metabolism and AMPK pathway activation , which should be considered when interpreting results.