PIK3C3 Antibody

What is PIK3C3 and why is it important in cellular research?

PIK3C3 (Phosphatidylinositol 3-kinase catalytic subunit type 3), also known as VPS34, functions as the catalytic subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex. This protein mediates the formation of phosphatidylinositol 3-phosphate (PI3P), which plays crucial roles in multiple membrane trafficking pathways .

PIK3C3 exists in two major complexes:

• PIK3C3-C1: Involved in autophagosome initiation

• PIK3C3-C2: Functions in autophagosome maturation and endocytosis

Its importance stems from its central role in:

• Autophagy regulation

• Endocytic trafficking

• Vesicle formation

• Lysosomal enzyme transport

Research applications targeting PIK3C3 are critical for understanding disorders including neurodegeneration, cancer, and autoimmune diseases .

What are the common applications for PIK3C3 antibodies in laboratory research?

PIK3C3 antibodies serve multiple research applications:

These applications enable researchers to investigate PIK3C3 expression patterns, localization changes under various conditions, and functional interactions with other proteins in autophagy and membrane trafficking pathways .

What tissue expression patterns does PIK3C3 exhibit and how should researchers account for this?

PIK3C3 demonstrates variable expression across human tissues:

High expression tissues:

• Ovary, adrenal gland, and colon

• Testes, rectum, and certain immune cells (monocytes, CD4+ T cells)

• Cerebellum, spinal cord, and various brain regions

Methodological considerations:

• Include appropriate positive control tissues (cerebellum, ovary) in experimental designs

• Account for tissue-specific expression levels when comparing results across tissue types

• Use multiple detection methods to confirm expression patterns

• Consider tissue-specific cofactors that may affect antibody binding

When studying PIK3C3 in tissues with lower expression, more sensitive detection methods and careful validation are required to avoid false negative results.

What are the critical validation steps when using PIK3C3 antibodies?

Proper validation requires:

• Specificity testing:

  • Positive/negative tissue controls known to express/not express PIK3C3

  • Peptide competition assays with immunizing peptide

  • Knockdown/knockout validation (siRNA or CRISPR)

• Application-specific validation:

  • For Western blot: Verify molecular weight (101.5 kDa)

  • For IHC/IF: Compare staining pattern with known subcellular localization

• Cross-reactivity assessment:

  • Test on predicted cross-reactive species when studying non-human models

  • Validate specificity in species of interest, especially for polyclonal antibodies

• Epitope considerations:

  • Document the specific epitope region (e.g., AA 14-39, AA 150-250, AA 770-801)

  • Consider whether post-translational modifications might affect antibody binding

How should PIK3C3 antibodies be stored and handled to maintain reactivity?

Optimal storage and handling protocols:

Storage conditions:

• Short-term (up to 2 weeks): Refrigerate at 2-8°C

• Long-term: Store at -20°C in small aliquots to prevent freeze-thaw cycles

Handling precautions:

• Avoid repeated freeze-thaw cycles which degrade antibody quality

• When using glycerol-containing formulations, centrifuge briefly before opening

• Reconstitute lyophilized antibodies completely before use

• Allow antibodies to reach room temperature before opening containers

Working solution preparation:

• Dilute in appropriate buffer systems with stabilizing proteins

• For immunofluorescence applications, prepare fresh working solutions

• Consider adding sodium azide (0.09% W/V) to prevent microbial contamination in stored solutions

How do different PIK3C3 antibody epitopes affect experimental outcomes in autophagy research?

Different epitope-targeting antibodies provide distinct experimental advantages:

Methodological considerations:

• For autophagy studies, N-terminal antibodies may better detect total PIK3C3 levels

• When studying PIK3C3 complexes, epitope accessibility may be compromised in certain protein interactions

• Using antibodies targeting different regions can provide complementary data and confirm structural observations

• Phospho-specific antibodies are essential for studying regulatory mechanisms but require careful protocol optimization

What is the role of PIK3C3 in autoimmune disease pathogenesis and how can antibodies help investigate this connection?

PIK3C3 shows significant involvement in autoimmune pathology:

• Multiple sclerosis model findings:

  • PIK3C3 deficiency in myeloid cells imparts partial resistance to experimental autoimmune encephalomyelitis (EAE)

  • This protection correlates with reduced CD4+ T cell accumulation in the CNS

  • PIK3C3 inhibition using SAR405 delays disease progression

• Systemic lupus erythematosus connections:

  • PIK3C3 promoter variant (rs3813065/-442 C/T) associates with autoantibody profiles

  • Strong association with simultaneous anti-Ro and anti-Sm antibodies in African-American patients

  • Links to differential expression of peptide processing enzymes

Research applications of PIK3C3 antibodies:

• Monitor PIK3C3 expression in immune cell subsets during disease progression

• Assess autophagy activity in myeloid cells using co-staining with autophagy markers

• Evaluate effects of PIK3C3 inhibitors on inflammatory cytokine production

• Investigate PIK3C3-dependent IL-1β production in myeloid cells

How does PIK3C3 function in cancer development, and what technical approaches with antibodies best study these mechanisms?

PIK3C3 demonstrates complex roles in cancer biology:

• Cancer-related functions:

  • Upregulated in hepatocellular carcinoma tissues and cancer stem cells

  • Facilitates liver cancer stem cell expansion

  • Functions both through autophagy-dependent and independent mechanisms

  • PIK3C3 inhibition blocks cancer stem cell expansion induced by PI3K inhibitors

• Technical approaches with antibodies:

  • Flow cytometry: Combine PIK3C3 antibodies with cancer stem cell markers for population analysis

  • Proximity ligation assays: Investigate PIK3C3 interactions with other proteins in cancer signaling

  • Multiplexed immunofluorescence: Study PIK3C3 colocalization with organelle markers in tumor sections

  • Live-cell imaging: Monitor PIK3C3 dynamics with tagged antibody fragments in cancer cell models

• Methodological considerations:

  • Use both phospho-specific and total PIK3C3 antibodies to assess activation state

  • Combine with autophagy markers (LC3, p62) to differentiate autophagy-dependent vs. independent functions

  • Consider dual inhibition strategies (PIK3C3 + PI3K) for cancer research applications

What are the challenges in detecting phosphorylated forms of PIK3C3 and how can these be overcome?

Detecting phosphorylated PIK3C3 presents specific challenges:

• Common technical difficulties:

  • Low abundance of phosphorylated forms

  • Rapid dephosphorylation during sample preparation

  • Epitope masking in protein complexes

  • Insufficient specificity of phospho-specific antibodies

• Methodological solutions:

  • Enhanced sample preservation: Include phosphatase inhibitors in all buffers

  • Enrichment techniques: Use phospho-protein enrichment kits before immunoprecipitation

  • Signal amplification: Employ tyramide signal amplification for IHC/IF applications

  • Validation controls: Include samples treated with phosphatases as negative controls

• Application-specific optimizations:

  • For Western blotting: Use PVDF membranes and optimize blocking conditions

  • For immunoprecipitation: Consider sequential IP approaches

  • For immunofluorescence: Increase antibody concentration and extend incubation times

• Key phosphorylation sites:

  • pSer164: Associated with regulatory functions

  • pSer282: Important for protein-protein interactions

What are common causes of false negative/positive results when using PIK3C3 antibodies, and how can they be addressed?

False negatives:

• Epitope masking in protein complexes

  • Solution: Try antibodies targeting different epitopes

  • Use denaturing conditions when appropriate

• Insufficient antigen retrieval in IHC

  • Solution: Optimize antigen retrieval methods (heat, pH)

  • Extend retrieval time for formalin-fixed tissues

• Degradation during sample preparation

  • Solution: Use protease inhibitors

  • Process samples at 4°C to minimize degradation

False positives:

• Cross-reactivity with related PI3K family members

  • Solution: Validate with knockout/knockdown controls

  • Use epitope-specific blocking peptides

• Non-specific binding in high-expressing tissues

  • Solution: Titrate antibody concentration

  • Include appropriate negative controls

• Secondary antibody cross-reactivity

  • Solution: Use isotype-specific secondary antibodies

  • Include secondary-only controls

How can researchers distinguish between PIK3C3's autophagy-dependent and autophagy-independent functions in experimental settings?

Differentiating these functions requires strategic experimental approaches:

• Complementary genetic approaches:

  • Compare PIK3C3 inhibition/knockdown with knockdown of autophagy-specific proteins (ATG5, ATG7)

  • Effects present with PIK3C3 manipulation but absent in ATG protein manipulation suggest autophagy-independent functions

• Pharmacological strategy:

  • Compare PIK3C3-specific inhibitors with broader autophagy inhibitors

  • Use time-course studies to separate early (often autophagy-independent) from late (autophagy-dependent) effects

• Methodological applications:

  • Multiplex immunofluorescence with LC3 and PIK3C3 antibodies to assess colocalization

  • Combine with transmission electron microscopy to visualize autophagosome formation

  • Monitor PIK3C3 kinase activity using specialized PI3P biosensors alongside functional readouts

• Context-specific considerations:

  • In cancer studies, monitor both autophagy markers and non-autophagic targets simultaneously

  • In immune cells, assess effects on endocytic trafficking separately from autophagy

How are PIK3C3 antibodies being used to study the connections between autophagy and metabolic regulation?

Recent research reveals emerging applications:

• Adipose tissue metabolism:

  • PIK3C3 regulates white adipose tissue function and autophagy

  • Adipose-specific PIK3C3 knockout mice show altered ER stress responses

  • Age-dependent effects on glucose tolerance and adiposity

  • PIK3C3 antibodies help track these metabolic changes through tissue analysis

• T cell metabolism investigations:

  • PIK3C3 deficiency impairs T cell metabolism and mitochondrial activity

  • Antibody-based detection reveals reduced active mitochondria upon T cell activation

  • Multiplex approaches combining PIK3C3 antibodies with metabolic markers provide insights

• Technical innovations:

  • Combining metabolic flux analysis with PIK3C3 immunodetection

  • Spatially resolved proteomics with PIK3C3 antibodies to identify metabolic microenvironments

  • Live-cell metabolic imaging with PIK3C3 detection

• Translational implications:

  • PIK3C3 antibodies help identify therapeutic windows for metabolic disease intervention

  • Dual-targeting of PIK3C3 and metabolic pathways may have synergistic effects in disease models

What considerations should researchers make when selecting PIK3C3 antibodies for multi-omics integration studies?

Multi-omics integration requires careful antibody selection:

• Compatibility with sample processing:

  • Ensure antibody compatibility with fixation methods needed for spatial transcriptomics

  • Consider epitope preservation in protocols requiring harsh extraction methods

• Technical integration factors:

  • Choose antibodies validated for multiplexed applications (CyTOF, multiplexed IF)

  • Select clones with documented cross-platform consistency

• Data normalization approaches:

  • Include universal controls across experimental platforms

  • Consider using multiple antibodies targeting different PIK3C3 epitopes to strengthen data integration

• Application-specific recommendations:

  • For proteogenomic integration: Select antibodies validated against recombinant protein standards

  • For spatial biology applications: Choose antibodies with demonstrated specificity in tissue contexts

  • For single-cell approaches: Validate antibodies specifically for low-input applications