PLA2G4C Antibody

What is PLA2G4C and why is it significant in research?

PLA2G4C (Phospholipase A2, Group IVC) is a calcium-independent phospholipase, lysophospholipase, and O-acyltransferase involved in phospholipid remodeling. Its importance stems from its role in:

• Endoplasmic reticulum membrane homeostasis and lipid droplet biogenesis

• Preferential hydrolysis of the ester bond of fatty acyl groups at the sn-2 position of phospholipids

• Participation in deacylation-reacylation cycles of membrane phospholipids

• Transfer of sn-1 fatty acyl from one lysophospholipid molecule to the sn-2 position of another

This enzyme has gained significant research interest due to its involvement in Hepatitis C virus (HCV) replication and assembly, where it contributes to the formation of membranous webs essential for viral replication .

What types of PLA2G4C antibodies are available for research and what are their characteristics?

Several types of PLA2G4C antibodies are available with varying properties:

Most commercially available antibodies are purified using antigen affinity chromatography and provided in buffer containing phosphate-buffered saline with preservatives such as sodium azide and stabilizers like glycerol .

How should researchers optimize Western blot protocols when using PLA2G4C antibodies?

For optimal Western blot results with PLA2G4C antibodies:

• Sample preparation: PLA2G4C has low endogenous expression in some cell lines (e.g., Huh7.5.1 cells), requiring enrichment through immunoprecipitation before detection . Standard cell lysates work well for Jurkat and HeLa cells .

• Dilution optimization:

  • Start with dilutions between 1:500-1:2000 for Western blotting

  • For low expression samples, consider using more concentrated antibody (1:500) and longer exposure times

• Expected molecular weight: Look for bands at approximately 60-72 kDa (the calculated molecular weight is ~61 kDa, but observed weight may be higher due to post-translational modifications)

• Validation controls: Include positive control lysates from Jurkat or HeLa cells as demonstrated in validation data

• Signal enhancement: For samples with low PLA2G4C expression, consider using high-sensitivity detection reagents or signal amplification systems

For detection of subtle changes in expression, as seen in HCV infection studies, longer exposure times might be necessary as PLA2G4C expression is upregulated upon HCV infection but remains at relatively low levels .

What are the optimal conditions for immunohistochemistry using PLA2G4C antibodies?

When performing immunohistochemistry with PLA2G4C antibodies:

• Tissue preparation and antigen retrieval:

  • For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval with TE buffer pH 9.0 is recommended

  • Alternatively, citrate buffer pH 6.0 can be used

  • Fresh preparation of fixatives (PFA) is recommended as long-term stored PFA can convert to formalin, affecting results

• Antibody dilution:

  • Use dilutions between 1:100-1:400 for IHC applications

  • Titrate in each testing system to determine optimal concentration

• Detection systems:

  • Both chromogenic and fluorescent secondary detection systems are compatible

  • For low expression, consider using amplification systems like tyramide signal amplification

• Positive control tissues:

  • Human heart tissue and human skeletal muscle tissue have been validated as positive controls

  • Include these tissues alongside experimental samples to verify staining protocols

• Blocking and washing steps:

  • Thorough blocking with appropriate serum (typically 5-10% normal serum from the species of the secondary antibody)

  • Extend washing steps (3-5x for 5 minutes each) to reduce background

Validation of antibody specificity using peptide blocking experiments is recommended, especially for tissues not previously characterized for PLA2G4C expression .

How does PLA2G4C contribute to HCV replication and what methodologies can detect this interaction?

PLA2G4C plays multiple critical roles in the HCV life cycle:

• Membranous web (MW) formation: PLA2G4C is essential for the formation of the membranous web, a remodeled intracellular structure where HCV replicates its genome. Electron microscopy revealed that siRNA-mediated knockdown of PLA2G4C reduced MW structures from 67% to only 13% of Lunet-Con1 cells .

• HCV replication: Knockdown of PLA2G4C by siRNA significantly suppressed HCV RNA replication. Chemical inhibition with methyl arachidonyl fluorophosphonate (MAFP) also reduced HCV replication .

• HCV assembly: PLA2G4C transports HCV nonstructural proteins from replication sites to lipid droplets, facilitating virion assembly .

For detection of these interactions, researchers can employ:

• Co-immunoprecipitation: PLA2G4C antibodies can be used to examine interactions with viral proteins (NS4B, NS5A)

• Immunofluorescence confocal microscopy: To visualize colocalization of PLA2G4C with HCV proteins

• Electron microscopy: To examine MW structure formation

• siRNA-mediated knockdown: Combined with PLA2G4C antibody detection to verify knockdown efficiency

An experimental approach would include:

• Transfection of cells with siRNA targeting PLA2G4C

• Infection with HCV (e.g., JFH-1 strain)

• Western blot analysis using PLA2G4C antibodies to confirm knockdown

• Quantitative RT-PCR to measure HCV RNA levels

• Immunofluorescence and electron microscopy to assess MW formation

How is PLA2G4C expression altered in inflammatory diseases and what methodologies can accurately quantify these changes?

PLA2G4C expression shows distinct patterns in inflammatory conditions like asthma:

• Expression changes in asthma:

  • Increased PLA2G4C expression observed in PBMCs from severe asthmatics compared to non-severe asthmatics and healthy controls

  • Upregulation is further enhanced upon stimulation with allergens (Der p1) or LPS

• Methodological approaches for quantification:

  a) mRNA quantification:

  • qRT-PCR with appropriate housekeeping genes for normalization

  • Results should be presented as fold-change relative to healthy controls

  b) Protein detection:

  • Western blot using validated PLA2G4C antibodies

  • Proper normalization with housekeeping proteins

  • Consider enrichment through immunoprecipitation for low-expression samples

  c) Statistical analysis:

  • Principal Component Analysis (PCA) can reveal correlations between PLA2G4C and other inflammatory markers

  • This approach demonstrated that PLA2G4C and PLA2G12 expression patterns differentiate between allergen and LPS responses

When designing experiments to study PLA2G4C in inflammatory conditions:

• Include appropriate disease controls (mild vs. severe cases)

• Test multiple stimuli (allergens, microbial products)

• Compare mRNA and protein expression levels

• Correlate with clinical parameters

• Consider cell-type specific expression patterns

What are common challenges when working with PLA2G4C antibodies and how can they be addressed?

Researchers working with PLA2G4C antibodies frequently encounter these challenges:

• Low endogenous expression:

  • Solution: Enrich PLA2G4C by immunoprecipitation before Western blot analysis

  • Alternative: Use cell lines with higher expression (e.g., Jurkat cells)

  • Consider using more sensitive detection methods or longer exposure times

• Background issues in immunostaining:

  • Solution: Optimize blocking with 5-10% serum from the species of secondary antibody

  • Increase washing steps (3-5x for 5 minutes each)

  • Use more dilute antibody concentrations after initial optimization

  • Consider using fluorescent detection which may offer better signal-to-noise ratio

• Cross-reactivity concerns:

  • Solution: Include appropriate negative controls

  • Validate antibody specificity using blocking peptides where available

  • Consider using multiple antibodies targeting different epitopes of PLA2G4C

• Species cross-reactivity:

  • Solution: Check sequence homology between target species

  • For untested species (e.g., monkey), perform validation studies with appropriate positive controls

  • Consider custom validation if working with non-standard research models

• BSA interference:

  • Solution: Request BSA-free formulations if needed for specific applications

  • For conjugation experiments, use antibodies without carriers

How can researchers design experiments to investigate PLA2G4C function in lipid metabolism and viral infection simultaneously?

When studying the dual role of PLA2G4C in lipid metabolism and viral infection:

• Experimental design strategy:

  a) Cell culture systems:

  • Use Huh7.5.1 cells which support both HCV replication and lipid droplet formation

  • Compare results in cells with and without HCV infection or replicons

  b) Genetic manipulation approaches:

  • siRNA knockdown (validated with siPLA2G4C-7 and siPLA2G4C-4)

  • Overexpression systems using tagged PLA2G4C constructs

  • CRISPR/Cas9 gene editing for complete knockout studies

  c) Chemical modulation:

  • Use methyl arachidonyl fluorophosphonate (MAFP) as a specific PLA2 inhibitor

  • Compare with structurally distinct PLA2 inhibitors to confirm specificity

• Readout methodologies:

  a) Viral replication analysis:

  • qRT-PCR for viral RNA quantification

  • Luciferase reporter systems for high-throughput studies

  • Focus-forming assays for infectious virus production

  b) Lipid metabolism assessment:

  • Lipid droplet visualization and quantification using fluorescent dyes

  • Lipidomics analysis of membrane phospholipid composition

  • Enzymatic activity assays for PLA2G4C

  c) Protein interaction studies:

  • Co-immunoprecipitation of PLA2G4C with viral proteins and lipid metabolism factors

  • Proximity ligation assays for in situ detection of protein-protein interactions

  • Live-cell imaging using fluorescently tagged proteins

• Validation approaches:

  • Use multiple antibodies targeting different PLA2G4C epitopes

  • Include appropriate controls (e.g., other PLA2 family members)

  • Rescue experiments in knockdown systems by expressing siRNA-resistant PLA2G4C

This comprehensive experimental approach would provide insights into how PLA2G4C simultaneously supports viral replication and modulates lipid metabolism, potentially identifying points of intervention that could disrupt viral replication without severely affecting normal cellular functions .

What emerging methodologies might enhance detection and functional analysis of PLA2G4C in complex biological systems?

Several emerging technologies hold promise for advancing PLA2G4C research:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize PLA2G4C localization at membranous webs and lipid droplets with nanometer precision

  • Correlative light and electron microscopy (CLEM) to connect fluorescent antibody labeling with ultrastructural context

  • Live-cell imaging using split fluorescent protein systems to monitor dynamic interactions

• Single-cell analysis approaches:

  • Single-cell RNA-seq to identify heterogeneity in PLA2G4C expression across cell populations

  • Mass cytometry with metal-conjugated PLA2G4C antibodies for high-dimensional phenotyping

  • Spatial transcriptomics to map PLA2G4C expression within tissue contexts

• Proximity-based interaction mapping:

  • BioID or APEX2 proximity labeling fused to PLA2G4C to identify the complete interactome

  • Split-BioID systems to map context-specific interactions

  • Förster resonance energy transfer (FRET) to monitor real-time interactions with binding partners

• CRISPR-based technologies:

  • CRISPRi for temporal control of PLA2G4C expression

  • Base editing for introducing specific mutations to probe structure-function relationships

  • CRISPR activation systems to upregulate endogenous PLA2G4C in models with low expression

• Nanobody and single-domain antibody development:

  • Development of high-affinity, small-format antibodies for improved tissue penetration

  • Intrabody applications to track and manipulate PLA2G4C in living cells

These methods would enhance our ability to study PLA2G4C's roles in membrane remodeling during both normal cellular processes and pathological conditions like viral infection.

How can researchers reconcile contradictory findings regarding PLA2G4C expression patterns across different disease models?

When faced with contradictory findings regarding PLA2G4C expression:

• Systematic comparative analysis:

  • Directly compare experimental protocols including cell types, stimuli, and detection methods

  • Standardize quantification approaches for both mRNA and protein measurements

  • Use absolute quantification methods (e.g., digital PCR, quantitative proteomics with labeled standards)

• Context-dependent expression analysis:

  • Investigate whether PLA2G4C expression is regulated differently in subpopulations within heterogeneous samples

  • Consider temporal dynamics - expression may vary during different disease stages

  • Examine potential post-translational modifications that affect antibody detection

• Comprehensive validation strategy:

  • Use multiple antibodies targeting different epitopes

  • Employ complementary techniques (e.g., mass spectrometry validation of Western blot findings)

  • Include genetic validation approaches (e.g., CRISPR knockout controls)

• Meta-analysis approach:

  • Create a standardized reporting format for PLA2G4C expression data

  • Compile findings across multiple studies with detailed metadata

  • Apply statistical methods to identify factors explaining divergent results

For example, research has shown that PLA2G4C expression is upregulated during HCV infection but not in cells containing only HCV replicons, suggesting that viral structural proteins are required for this regulation . In contrast, in asthma models, PLA2G4C expression is differentially regulated by allergens versus bacterial products (LPS) . These seemingly contradictory findings can be reconciled by recognizing the context-specific regulation of PLA2G4C expression.

A robust approach would involve parallel analysis of multiple disease models using standardized protocols and comprehensive reporting of experimental conditions to identify the mechanistic basis for differential expression patterns.