PANX3 Antibody, HRP conjugated

What is PANX3 and why is it important in inflammatory research?

PANX3 (Pannexin-3) is a membrane protein belonging to the pannexin family with critical functions in inflammation regulation. Recent studies have demonstrated that PANX3 plays a defensive role in dental pulp inflammation by inhibiting pro-inflammatory cytokines such as IL-1β and IL-6. Immunohistochemical analysis reveals that PANX3 is primarily expressed in the odontoblast layer of normal dental pulp tissue, with significantly diminished levels at inflammatory sites in both human and rat dental pulp tissues . PANX3 is expressed in both the cell membrane and cytoplasm, functioning through various cellular mechanisms to modulate inflammatory responses. Its protective role makes it a potential therapeutic target for inflammatory conditions, particularly in dental pulp inflammation.

What are the key specifications of PANX3 antibody, HRP conjugated?

The commercially available PANX3 antibody (HRP conjugated) has the following technical specifications:

This antibody has been generated against amino acids 135-215 of human PANX3, making it specifically useful for detecting human PANX3 in experimental systems .

How does HRP conjugation benefit PANX3 detection methodologies?

HRP (Horseradish Peroxidase) conjugation provides significant methodological advantages for PANX3 detection:

• Enhanced sensitivity: The enzymatic activity of HRP amplifies detection signals, enabling identification of low-abundance PANX3 protein in experimental samples.

• Direct detection capability: The conjugation eliminates the need for secondary antibody incubation, thereby streamlining experimental protocols and reducing potential sources of background noise.

• Flexible detection options: HRP conjugated antibodies can be used with various substrates (colorimetric, chemiluminescent, or fluorescent), offering versatility in experimental design and readout methods.

• Quantitative analysis potential: HRP provides a stable and reproducible signal that can be accurately measured for quantitative determination of PANX3 levels, particularly valuable in dose-response and time-course studies of PANX3 regulation.

• Compatibility with multiple fixation protocols: HRP conjugated antibodies typically perform well under various sample preparation conditions, making them adaptable to different experimental contexts.

For PANX3 research specifically, the HRP conjugation enhances detection sensitivity in ELISA applications, the primary validated application for this antibody .

What are the optimal storage and handling conditions for PANX3 antibody?

To maintain optimal activity of the PANX3 antibody (HRP conjugated), researchers should adhere to the following storage and handling practices:

• Storage temperature: The antibody should be aliquoted and stored at -20°C to maintain long-term stability .

• Avoid freeze/thaw cycles: Repeated freezing and thawing significantly reduces antibody activity and should be minimized by preparing appropriately sized aliquots .

• Buffer composition: The antibody is supplied in a formulation containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol. The high glycerol content helps prevent freezing damage during storage .

• Light protection: As an HRP-conjugated antibody, protection from prolonged light exposure is advisable to prevent potential photobleaching of the conjugate.

• Working solution stability: When preparing working dilutions, use freshly prepared solutions when possible. If storage is necessary, add protein stabilizers (e.g., BSA) and keep at 4°C for short-term use only.

• Temperature during handling: Maintain samples on ice when working with the antibody to preserve enzymatic activity of the HRP conjugate.

• Contamination prevention: Use sterile technique when handling antibody solutions to prevent microbial growth that could degrade the antibody or introduce experimental artifacts.

How can PANX3 antibody be utilized to investigate TNF-α-mediated regulation?

The relationship between PANX3 and TNF-α represents a significant area of investigation that can be methodically explored using the PANX3 antibody through the following approaches:

• Dose-response analysis: Research has demonstrated that TNF-α downregulates PANX3 expression in a concentration-dependent manner. Treatment of human dental pulp cells (HDPCs) with increasing concentrations of TNF-α (0, 0.1, 1, 10 ng/ml) for 24 hours progressively reduces PANX3 mRNA expression by 25%, 30%, and 70%, respectively . The PANX3 antibody can be used to confirm these findings at the protein level through ELISA quantification.

• Pathway dissection experiments: Pre-treatment with specific inhibitors before TNF-α stimulation reveals mechanistic insights:

  • Proteasome inhibitor (MG132, 1 μM, 30 min pre-treatment) partially rescues TNF-α-induced PANX3 downregulation, indicating proteasomal degradation as a key mechanism .

  • NF-κB inhibitor (BAY 11-7082, 2 μM, 30 min pre-treatment) unexpectedly exacerbates PANX3 inhibition rather than reversing it, suggesting complex pathway interactions .

• Time-course monitoring: Using the PANX3 antibody in ELISA to track temporal changes in PANX3 protein levels following TNF-α stimulation can reveal the kinetics of this regulatory relationship.

• Subcellular localization analysis: Since PANX3 exhibits both membrane and cytoplasmic expression, investigating whether TNF-α differentially affects these PANX3 pools provides deeper mechanistic understanding.

This systematic approach reveals that TNF-α regulates PANX3 through proteasomal degradation rather than transcriptional inhibition, informing experimental design for inflammatory response studies .

What protocols are recommended for investigating PANX3's role in inflammatory signaling?

When exploring PANX3's function in inflammatory signaling cascades, the following methodological approaches are recommended:

• Genetic manipulation studies:

  • PANX3 overexpression: Transfection with lentiviral systems harboring PANX3 cDNA has demonstrated significant suppression of TNF-α-induced pro-inflammatory cytokines (IL-1β and IL-6) .

  • PANX3 knockdown: Lentiviral shRNA targeting PANX3 results in exacerbated expression of pro-inflammatory cytokines following TNF-α stimulation .

  • Verification of manipulation: The PANX3 antibody is essential for confirming successful overexpression or knockdown through ELISA or other detection methods.

• NF-κB pathway analysis:

  • Dual-luciferase reporter assay: PANX3 overexpression decreases NF-κB activation by approximately 60%, while knockdown increases activity 1.7-fold compared to controls .

  • Western blot analysis: PANX3 overexpression decreases phosphorylated-p65(S536) expression and suppresses IκBα degradation following TNF-α stimulation (30 min) .

  • Immunofluorescence assay: Nuclear translocation of p65 is increased in PANX3 knockdown cells upon TNF-α stimulation .

• Recovery experiments:

  • Pre-treatment with NF-κB inhibitor (BAY 11-7082) attenuates the exaggerated effects of PANX3 knockdown on TNF-α-induced inflammatory cytokine expression, confirming pathway dependency .

• Quantification protocols:

  • qRT-PCR and ELISA for cytokine measurement

  • Phospho-specific antibody detection for signaling pathway activation

  • Time-course designs to capture signaling dynamics

These findings collectively demonstrate that PANX3 negatively regulates NF-κB signaling, establishing a molecular mechanism by which PANX3 exerts its anti-inflammatory effects .

How can PANX3 antibody help resolve contradictory data in inflammation research?

Research on PANX3 presents an apparent paradox: PANX3 levels decrease during inflammation, yet experimental PANX3 overexpression suppresses inflammatory responses. The PANX3 antibody can help resolve such contradictions through:

• Context-dependent expression analysis:

  • Systematically quantify PANX3 levels across:

    • Different cell types (odontoblasts, fibroblasts, immune cells)

    • Various inflammatory stimuli (TNF-α, IL-1β, LPS)

    • Diverse tissue contexts (dental pulp, joints, vascular tissues)

  • Compare expression patterns between in vitro models and clinical samples

• Temporal resolution studies:

  • Conduct detailed time-course experiments using PANX3 antibody-based ELISA

  • Correlate PANX3 levels with inflammatory markers at multiple time points

  • Research shows dynamic regulation where initial PANX3 downregulation may be followed by compensatory mechanisms

• Mechanistic dissection:

  • Dual-luciferase reporter assays reveal that PANX3 suppresses NF-κB activity by approximately 60%, providing a mechanism for its anti-inflammatory effects .

  • Analysis of the Panx3 promoter using Genomatix MatInspector software indicates three putative NF-κB binding sites, suggesting that NF-κB might actually promote PANX3 expression as part of a negative feedback loop .

  • This creates a complex regulatory circuit where TNF-α initially decreases PANX3 through proteasomal degradation, but NF-κB activation may eventually restore PANX3 levels to limit excessive inflammation.

• Negative feedback hypothesis testing:

  • The data suggests PANX3 participation in a negative feedback loop where:

    • Initial inflammation triggers PANX3 degradation, permitting inflammatory response

    • Subsequent NF-κB activation may upregulate PANX3 expression

    • Increased PANX3 then inhibits NF-κB activity, limiting inflammation

This complex regulatory relationship explains seemingly contradictory findings and positions PANX3 as a potential therapeutic target for modulating inflammatory responses .

What controls are essential when studying PANX3 degradation mechanisms?

When investigating PANX3 degradation pathways, particularly the proteasomal mechanism identified in TNF-α stimulation, the following controls are essential:

• Pathway-specific inhibitor controls:

  • Proteasome inhibitor: MG132 (1 μM) partially rescues TNF-α-induced PANX3 degradation, confirming proteasomal involvement .

  • NF-κB inhibitor: BAY 11-7082 (2 μM) exacerbates PANX3 reduction, indicating complex pathway interactions .

  • Vehicle control: DMSO at equivalent concentration to inhibitors

  • Alternative pathway inhibitors: Lysosomal inhibitors to rule out autophagy-dependent degradation

• Time-course controls:

  • Pre-treatment timing: 30 minutes before TNF-α stimulation has been validated as effective .

  • Duration of TNF-α exposure: Both acute (30 min) and extended (24 hr) timepoints should be examined to distinguish immediate from secondary effects.

  • Sequential sampling: Capture the dynamics of PANX3 degradation with multiple timepoints.

• Concentration gradient controls:

  • TNF-α dose-response: 0.1, 1, and 10 ng/ml have been validated, with maximum effect at 10 ng/ml .

  • Inhibitor concentration optimization: Test multiple concentrations to establish dose-dependent effects.

• Protein synthesis controls:

  • Cycloheximide chase experiments to distinguish degradation from synthesis inhibition

  • Actinomycin D to block transcription and isolate post-transcriptional regulation

• Molecular verification controls:

  • Ubiquitination analysis: Immunoprecipitation followed by ubiquitin detection

  • Proteasome activity assays to confirm inhibitor efficacy

  • PANX3 mutants lacking potential ubiquitination sites

The research data indicates that TNF-α reduces PANX3 protein levels via proteasomal degradation, and this effect can be partially reversed by MG132 . These controls help establish the specificity and mechanism of this regulatory pathway.

How can PANX3 antibody be optimized for studying PANX3 interactions with potential binding partners?

To investigate PANX3 interactions with potential binding partners, researchers should consider the following methodological optimizations:

• Co-immunoprecipitation approach:

  • For immunoprecipitation, unconjugated PANX3 antibody may be preferable to HRP-conjugated versions to prevent steric hindrance.

  • Membrane protein-specific lysis buffers containing mild detergents (e.g., digitonin, CHAPS) are recommended as PANX3 is membrane-associated.

  • Crosslinking approaches may help capture transient interactions.

• Potential interaction partners to investigate:

  • NF-κB pathway components: Research shows PANX3 regulates NF-κB activation, suggesting potential direct or indirect interactions .

  • BCL6: This transcriptional repressor has been identified as a potential PANX3 interaction partner that could mediate NF-κB suppression .

  • Ubiquitination machinery: Given PANX3's proteasomal degradation following TNF-α stimulation, investigating interactions with E3 ligases is warranted .

• Validation strategies:

  • Reciprocal co-immunoprecipitation with antibodies against suspected binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Subcellular co-localization studies using immunofluorescence

• Functional verification:

  • Mutation of potential interaction domains to disrupt binding

  • Competition assays with peptide fragments

  • In vitro binding assays with recombinant proteins

• Pathway-specific considerations:

  • The research suggests two potential mechanisms for PANX3-mediated NF-κB inhibition:

    • Interaction with BCL6, which can suppress NF-κB activity

    • Modulation of cAMP/PKA signaling, as PANX3 overexpression may decrease cAMP/PKA activity, potentially inhibiting p65 phosphorylation at Ser276

These approaches can help elucidate the molecular mechanisms by which PANX3 exerts its anti-inflammatory effects, particularly its negative regulation of NF-κB signaling.

What are the key considerations for validating PANX3 antibody in different applications?

While the PANX3 antibody (HRP conjugated) is validated for ELISA applications, researchers may want to extend its use to other techniques. Consider these validation approaches:

• Specificity validation:

  • Positive controls: Use samples with confirmed PANX3 expression (e.g., normal dental pulp tissue showing odontoblast layer expression) .

  • Negative controls: Use PANX3 knockdown cells or tissues with minimal PANX3 expression (e.g., inflamed dental pulp) .

  • Peptide competition assay: Pre-incubation with immunogen peptide (amino acids 135-215) should abolish specific binding .

• Application-specific optimization:

  • Western blot: Determine optimal sample preparation, blocking conditions, and antibody dilution.

  • Immunofluorescence: Optimize fixation methods, considering PANX3's dual localization in membrane and cytoplasm .

  • Flow cytometry: Develop appropriate permeabilization protocols for detecting cytoplasmic PANX3.

• Sensitivity assessment:

  • Prepare serial dilutions of recombinant PANX3 protein to establish detection limits

  • Compare sensitivity across applications to guide experimental design

• Cross-validation:

  • Compare results with alternative detection methods (e.g., qRT-PCR for mRNA expression)

  • Use multiple antibodies targeting different PANX3 epitopes when possible

• Reproducibility testing:

  • Repeat experiments across different lots of antibody

  • Document inter-assay and intra-assay variation

These validation steps ensure reliable and interpretable results when extending the use of PANX3 antibody beyond its validated ELISA application.

How should researchers interpret PANX3 expression changes in inflammatory models?

When analyzing PANX3 expression changes in inflammatory contexts, consider these interpretive guidelines:

• Baseline contextualization:

  • PANX3 is expressed in the odontoblast layer of normal dental pulp tissue .

  • Expression is significantly diminished at inflammatory sites in both human and rat dental pulp tissues .

  • TNF-α downregulates PANX3 expression in a dose-dependent manner, with maximum effect at 10 ng/ml .

• Temporal considerations:

  • Acute versus chronic inflammation may produce different PANX3 expression patterns

  • Research suggests dynamic regulation where initial downregulation may be followed by compensatory mechanisms

• Pathway integration:

  • PANX3 reduction by TNF-α occurs via proteasomal degradation rather than transcriptional inhibition .

  • NF-κB may actually promote PANX3 expression based on putative binding sites in the PANX3 promoter .

  • This creates a potential negative feedback loop where initial inflammation decreases PANX3, but subsequent NF-κB activation may restore levels.

• Functional correlation:

  • PANX3 overexpression suppresses pro-inflammatory cytokines (IL-1β, IL-6) .

  • PANX3 knockdown exacerbates inflammatory responses .

  • PANX3 negatively regulates NF-κB signaling pathway .

• Therapeutic implications:

  • The protective role of PANX3 in inflammation suggests potential therapeutic strategies

  • Stabilizing PANX3 against degradation might provide anti-inflammatory benefits

  • Understanding the temporal dynamics of PANX3 regulation could inform optimal intervention timing

This interpretive framework helps researchers contextualize their findings within the broader understanding of PANX3's role in inflammatory processes.