PLA2G4D Antibody

What is PLA2G4D and what specific domains should antibodies target for optimal detection?

PLA2G4D (Cytosolic phospholipase A2 delta) belongs to the phospholipase A2 enzyme family and catalyzes the hydrolysis of glycerophospholipids at the sn-2 position, liberating free fatty acids and lysophospholipids . When selecting antibodies, consider targeting conserved regions or specific functional domains:

• The N-terminal region (amino acids 1-216) is commonly used as an immunogen

• The region spanning amino acids 531-580 is also effective for antibody development

• For functional studies, antibodies targeting the catalytic domain containing the active site serine (S370) may be particularly useful

Antibody selection should be informed by your specific research question - whether you need to detect total protein, specific phosphorylation states, or distinguish between active and inactive forms.

Based on validated reactivity data, consider the following positive controls:

Note that PLA2G4D is poorly detectable in commonly used cell lines but is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines . For Western blot controls, transfected cells expressing recombinant PLA2G4D serve as reliable positive controls, showing bands at approximately 92 kDa .

How is PLA2G4D involved in the pathogenesis of psoriasis, and what methodological approaches are best for studying this connection?

PLA2G4D plays a critical role in psoriasis pathogenesis through a novel lipid antigen presentation pathway:

• Mechanism: IFN-α induces mast cells to release exosomes containing PLA2G4D, which transfers cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This leads to the generation of neolipid antigens recognized by lipid-specific CD1a-reactive T cells, inducing production of IL-22 and IL-17A .

• Methodological approaches:

  • T cell response assays: Measure PLA2G4D-dependent CD1a-reactive T cell responses in psoriasis patients versus healthy controls

  • Immunohistochemistry: Use PLA2G4D antibodies (1:100-1:400 dilution) with TE buffer pH 9.0 for antigen retrieval to detect expression in psoriatic skin lesions

  • Exosome isolation: Ultracentrifugation protocols to study PLA2G4D-containing exosomes from mast cells

  • Flow cytometry: To analyze CD1a-reactive T cells in patient samples

Research findings show significantly greater PLA2G4D-dependent CD1a-reactive T cell responses in psoriasis patients compared with healthy donors, suggesting PLA2 inhibition or CD1a blockade may have therapeutic potential .

What are the challenges in distinguishing PLA2G4D from other PLA2G4 family members, and how can these be overcome?

Distinguishing PLA2G4D from other phospholipase A2 family members (particularly PLA2G4A) presents several challenges:

• Structural similarities: PLA2G4D shares functional domains with other family members like PLA2G4A (cPLA2)

• Methodological solutions:

  • Specific antibodies: Use antibodies targeting unique regions of PLA2G4D (e.g., amino acids 531-580) that have minimal cross-reactivity with other family members

  • Western blot differentiation: PLA2G4D is observed at 92 kDa, while PLA2G4A typically appears at 85-100 kDa

  • Gene silencing controls: Include siRNA or knockout controls to verify antibody specificity

  • Recombinant protein standards: Use purified proteins as migration standards

  • Mass spectrometry validation: For definitive identification of the specific isoform

When publishing results, clearly document the specific antibody clone, epitope region, and validation methods used to distinguish between family members.

What protocols and considerations are important for studying PLA2G4D's calcium-dependent membrane interactions?

PLA2G4D interacts with membranes in a calcium-dependent manner , requiring specific methodological considerations:

• Subcellular fractionation protocol:

  • Lyse cells in 60 mM sodium phosphate buffer (pH 7.4) containing 0.1% NP-40 and protease inhibitors

  • Sonicate using a Sonopuls ultrasonic homogenizer (3 min with 0.5 s on/off cycle; amplitude 30%)

  • Centrifuge at 1,000 g to separate membrane and cytosolic fractions

• Calcium dependence assays:

  • Test enzyme activity with substrate solutions containing 1 mM of emulsified lipid supplemented with 2% FA-free BSA in PBS (pH 7.4)

  • Compare activity in the presence and absence of 1 mM CaCl₂

  • Use EGTA as a calcium chelator in control experiments

• Imaging approaches:

  • Confocal imaging of cells expressing tagged PLA2G4D to visualize translocation

  • Time-lapse microscopy before and after calcium ionophore treatment

For meaningful results, experiments should include both calcium-free and calcium-supplemented conditions, with appropriate positive controls (PLA2G4A) and negative controls (calcium-independent PLA2 family members).

How can site-directed mutagenesis be used to study PLA2G4D's active site, and what are the key residues to target?

Site-directed mutagenesis is a powerful approach for studying PLA2G4D's active site, with the serine-370 residue being particularly important:

• Mutagenesis protocol for S370A mutation:

  • Use Q5 site-directed mutagenesis kit with primers:

    • Forward: 5′-CAGTGGCATCgcgGGCTCTACAT-3′

    • Reverse: 5′-AAGTAGGTCACACAGTCC-3′

  • Set annealing temperatures according to NCBI primer tool

  • Use elongation time of 3:30 min (~20-30 s/kb)

  • Transform into chemically competent E. coli

  • Verify the base-exchange by Sanger sequencing

• Activity comparisons:

  • Express wild-type and mutant proteins

  • Compare enzymatic activities using in vitro assays

  • Measure both hydrolase and transacylase activities

• Key findings: Mutation of the active site serine to alanine (S370A) abolishes both hydrolase and transacylase activities, confirming the dual functionality of PLA2G4D as both a lipid hydrolase and transacylase .

What are the best methods for studying PLA2G4D in inflammatory conditions, especially in keratinocytes?

PLA2G4D expression is specifically upregulated in the upper epidermis of patients with inflammatory skin diseases , making methodological approaches for studying its role in inflammation particularly important:

• Cell culture models:

  • Primary human keratinocytes treated with proinflammatory cytokines (TNF-α, IL-17, IL-22)

  • 3D skin equivalents to model stratified epidermis

• Expression analysis approaches:

  • qRT-PCR for mRNA expression changes

  • Western blot with recommended dilutions (1:500-1:2000)

  • IHC of skin biopsies (1:100-1:400 dilution)

• Functional assays:

  • Lipidomic analysis to detect changes in lipid profiles

  • Transacylation activity assays using specific substrates

  • Compare wild-type and Pla2g4d-deficient mouse keratinocytes

• Transcriptomic analysis:

  • RNA-Seq to identify pathways modulated by PLA2G4D

  • Integration with lipidomic data for pathway analysis

Research findings demonstrate that Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, and transcriptomic analysis reveals that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling .

Why might I be detecting multiple bands or unexpected molecular weights when using PLA2G4D antibodies?

Multiple bands or unexpected molecular weights may occur for several reasons:

• Expected observations:

  • The calculated molecular weight of PLA2G4D is 92 kDa (818 amino acids)

  • Some antibodies report observed molecular weights of 60 kDa or 72 kDa

• Potential causes of discrepancies:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Proteolytic processing

  • Alternative splice variants

  • Cross-reactivity with other PLA2 family members

  • Sample preparation conditions (reducing vs. non-reducing)

• Optimization strategies:

  • Verify with recombinant protein control

  • Test different sample preparation methods

  • Perform peptide competition assays

  • Include knockout/knockdown controls

  • Try alternative antibody clones targeting different epitopes

Always document the specific bands observed and their molecular weights when reporting results, and include appropriate positive and negative controls to validate specificity.

What are the best approaches for improving detection of low-abundance PLA2G4D in tissue samples?

PLA2G4D is often expressed at low levels in many tissues but can be upregulated in inflammatory conditions :

• Sample enrichment techniques:

  • Immunoprecipitation before Western blotting

  • Cell fractionation to concentrate membrane-associated proteins

  • Cytokine stimulation (for in vitro samples) to upregulate expression

• Signal amplification methods:

  • Use high-sensitivity detection systems (ECL Prime, SuperSignal West Femto)

  • For IHC/IF: tyramide signal amplification

  • Consider polymer-based detection systems for IHC

• Antigen retrieval optimization for IHC:

  • Test both recommended methods: TE buffer pH 9.0 and citrate buffer pH 6.0

  • Optimize retrieval time and temperature

• Antibody selection tips:

  • For very low abundance targets, rabbit polyclonal antibodies may provide better sensitivity

  • Consider antibodies raised against full-length protein rather than peptides

When studying samples from inflammatory skin conditions, PLA2G4D detection is more likely to be successful due to its upregulation in these contexts .

How can I optimize experimental design to study both hydrolase and transacylase activities of PLA2G4D?

PLA2G4D exhibits dual functionality as both a lipid hydrolase and transacylase , requiring careful experimental design:

• In vitro activity assay protocol:

  • Incubate 1 μg of partially purified proteins with 20 μl substrate solution

  • Substrate solution: 1 mM of emulsified lipid supplemented with 2% FA-free BSA in PBS (pH 7.4)

  • Test both calcium-dependent (+1 mM CaCl₂) and calcium-independent conditions

  • Use S370A mutant as a negative control

• Substrate selection for different activities:

  • Hydrolase activity: Measure release of free fatty acids from phospholipids

  • Transacylase activity: Monitor transfer of acyl groups between lipid species

  • Include monoacylglycerols (MAGs) as potential acceptors to assess DAG formation

• Detection methods:

  • Thin-layer chromatography

  • Liquid chromatography-mass spectrometry (LC-MS)

  • Radioactive or fluorescently labeled lipids for high sensitivity

Research findings show that in the presence of exogenously added MAG, PLA2G4D strongly increases cellular DAG formation while MAG levels are decreased, confirming its transacylase activity .

How does the discovery of PLA2G4D's role in psoriasis change our approach to studying skin inflammatory diseases?

The identification of PLA2G4D's role in psoriasis represents a paradigm shift in understanding skin inflammatory diseases:

• Novel pathogenic mechanism:

  • PLA2G4D released via exosomes from mast cells transfers to CD1a-expressing cells

  • This leads to generation of neolipid antigens recognized by T cells

  • T cells then produce inflammatory cytokines IL-22 and IL-17A

• Research implications:

  • Focus on lipid antigen presentation pathways, not just protein antigens

  • Study CD1a-reactive T cells in addition to conventional T cells

  • Investigate exosome-mediated intercellular signaling in skin inflammation

• Therapeutic potential:

  • PLA2 inhibition as a novel therapeutic approach

  • CD1a blockade as an alternative strategy

  • Targeting exosome release from mast cells

These findings suggest an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to inflammation, opening new research directions and potential therapeutic targets .

What are the emerging techniques for studying PLA2G4D's interactions with lipid membranes?

Advanced techniques are enhancing our understanding of PLA2G4D's calcium-dependent membrane interactions:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM)

  • FRET-based assays to study protein-lipid interactions

  • Live-cell imaging with fluorescently tagged PLA2G4D

• Biophysical techniques:

  • Surface plasmon resonance to measure binding kinetics

  • Atomic force microscopy to visualize membrane interactions

  • Lipid bilayer models with controlled composition

• Computational approaches:

  • Molecular dynamics simulations of PLA2G4D-membrane interactions

  • Structural modeling of calcium-binding domains

  • Prediction of lipid binding sites

These methods provide deeper insights into the spatial and temporal dynamics of PLA2G4D's membrane interactions, which are essential for its enzymatic activity in both normal physiology and disease states.

How can omics approaches be integrated to better understand PLA2G4D's role in inflammatory processes?

Multi-omics integration offers comprehensive understanding of PLA2G4D's role in inflammation:

• Integrative approaches:

  • Lipidomics + Transcriptomics: Correlate lipid profile changes with gene expression

  • Proteomics + Interactomics: Identify PLA2G4D interaction partners

  • Single-cell multi-omics: Cell-specific expression and function

• Key findings from omics studies:

  • Transcriptomic analysis reveals PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling

  • Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment

• Methodological considerations:

  • Sample preparation must preserve lipid species

  • Targeted and untargeted approaches may be needed

  • Bioinformatic pipelines should integrate multiple data types

These integrated approaches can help elucidate the complex networks through which PLA2G4D influences inflammatory processes, potentially identifying new biomarkers and therapeutic targets.