COX2 Antibody

What is COX2 and why is it important in biomedical research?

COX2 (Cyclooxygenase-2), also known as Prostaglandin G/H synthase 2 (PGHS-2), is a key enzyme in the biosynthesis pathway of prostanoids derived from arachidonic acid. It possesses dual enzymatic functions: a cyclooxygenase activity that converts arachidonate to prostaglandin G2 (PGG2) and a peroxidase activity that reduces PGG2 to prostaglandin H2 (PGH2) . This process is central to the production of various prostaglandins and thromboxanes that mediate inflammatory responses.

Unlike COX1, which is constitutively expressed in most tissues, COX2 expression is typically low under normal physiological conditions but is rapidly upregulated during inflammation, following cellular stresses, and in response to growth factors, tumor promoters, hormones, bacterial endotoxins, and inflammatory cytokines such as Interleukin-1α . This inducible nature makes COX2 a critical target for anti-inflammatory drugs and cancer research, as its overexpression has been associated with various pathological conditions including cancer and inflammatory diseases .

Research on COX2 is particularly important because it represents a specific therapeutic target for non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX2 inhibitors (COXIBs), which are widely used clinically to manage pain and inflammation .

How do COX2 antibodies differ from COX1 antibodies in specificity and research applications?

COX2 antibodies are specifically designed to recognize epitopes unique to COX2 that are not present in COX1, despite the structural homology between these two isozymes. For example, the AS67 monoclonal antibody specifically binds to COX2 and does not cross-react with recombinant human COX1 protein as verified by immunoassay . This specificity is crucial for distinguishing between these closely related proteins in experimental settings.

In terms of research applications, COX2 antibodies are employed in various techniques including Western blotting (WB), immunocytochemistry/immunofluorescence (ICC/IF), immunoprecipitation (IP), and immunohistochemistry on paraffin sections (IHC-P) . The optimal antibody selection depends on the specific research application and experimental conditions.

COX2 antibodies are particularly valuable for studying inflammatory processes, cancer development, and treatment responses where COX2 expression is dynamically regulated, whereas COX1 antibodies might be more relevant for studying constitutive prostaglandin production in physiological contexts.

What are the recommended sample preparation protocols for detecting COX2 in different tissue types?

Sample preparation for COX2 detection varies based on the tissue type and experimental technique:

For Western blotting:

• Tissues should be homogenized in cold lysis buffer containing protease inhibitors to prevent COX2 degradation.

• Nuclear membrane fractions may require special attention as COX2 is located on the lumenal surface of the endoplasmic reticulum and on the inner and outer membranes of the nuclear envelope .

• Samples should be kept cold during preparation to prevent proteolysis.

For immunohistochemistry:

• Formalin fixation followed by paraffin embedding (FFPE) is compatible with many COX2 antibodies .

• Antigen retrieval methods (typically heat-induced in citrate buffer) are often necessary to expose epitopes masked during fixation.

• For tissues with high endogenous peroxidase activity, a hydrogen peroxide blocking step should be included.

For immunocytochemistry:

• Cells can be fixed with 4% paraformaldehyde and permeabilized with 0.1-0.5% Triton X-100.

• For optimal subcellular localization studies, membrane permeabilization conditions should be carefully optimized.

In all cases, proper controls should be included to validate antibody specificity, including positive controls (tissues known to express COX2, such as inflamed tissues) and negative controls (tissues where COX2 is not expected or COX2 knockout samples when available).

How can I optimize experimental conditions to study protein-protein interactions involving COX2?

Studying protein-protein interactions involving COX2 requires careful experimental design, as these interactions can significantly impact COX2 enzymatic activity through conformational changes or by affecting its subcellular localization . Several approaches can be optimized for this purpose:

Co-immunoprecipitation (Co-IP):

• Use antibodies specific to COX2 that are validated for immunoprecipitation applications .

• Employ gentle lysis conditions to preserve native protein complexes.

• Consider crosslinking approaches to stabilize transient interactions.

• Include appropriate negative controls (IgG or isotype controls) and positive controls (known interacting partners).

Proximity Ligation Assay (PLA):

• This technique allows visualization of protein-protein interactions in situ with high sensitivity.

• Use validated primary antibodies raised in different species against COX2 and its potential interacting partner.

• Optimize fixation and permeabilization conditions to maintain protein conformation while allowing antibody access.

Bimolecular Fluorescence Complementation (BiFC):

• Generate fusion constructs of COX2 and potential interacting proteins with split fluorescent protein fragments.

• Consider the orientation of fusion proteins to avoid steric hindrance of the interaction.

• Include appropriate controls to rule out non-specific complementation.

Recent research has identified several proteins that interact with COX2 and modulate its activity, including Fyn and ELMO1 . When designing experiments to study such interactions, consider that some may be dependent on COX2's enzymatic activity or specific posttranslational modifications.

What are the challenges in detecting posttranslational modifications of COX2, and how do they affect antibody recognition?

Detecting posttranslational modifications (PTMs) of COX2 presents several challenges:

• Specificity of antibodies against modified epitopes:

  • Antibodies recognizing specific PTMs (phosphorylation, glycosylation, s-nitrosylation) of COX2 must be highly specific to the modified residue .

  • Validation should include appropriate controls such as treatment with phosphatases or deglycosylating enzymes.

• Abundance of modified protein:

  • Often only a small fraction of total COX2 may be posttranslationally modified.

  • Enrichment strategies such as phosphoprotein or glycoprotein enrichment may be necessary before detection.

• Effects on antibody recognition:

  • PTMs can significantly alter epitope accessibility or conformation.

  • Some PTMs may mask epitopes recognized by certain antibodies, resulting in false negatives.

  • Conversely, some PTMs might create neo-epitopes that are recognized by antibodies not reactive to the unmodified protein.

• Methodological approach:

  • For phosphorylation studies, use phospho-specific antibodies combined with phosphatase treatment controls.

  • For glycosylation, consider enzymatic deglycosylation followed by mobility shift analysis.

  • For s-nitrosylation, the biotin-switch technique can be employed before immunodetection.

When investigating the functional significance of COX2 PTMs, it's important to note that these modifications can affect COX2 enzymatic activity, protein stability, subcellular localization, and protein-protein interactions . Therefore, experimental designs should incorporate approaches that can distinguish between these different functional outcomes.

How reliable are COX2 antibodies for detecting subcellular localization changes during cellular activation?

The reliability of COX2 antibodies for detecting subcellular localization changes depends on several factors:

• Antibody specificity and sensitivity:

  • Monoclonal antibodies like AS67 offer high specificity for COX2 over COX1 , which is crucial for distinguishing between these homologous proteins.

  • Different antibody clones may recognize different epitopes that might be differentially accessible depending on COX2's conformation or interactions in specific subcellular compartments.

• Fixation and permeabilization methods:

  • COX2 is located on the lumenal surface of the endoplasmic reticulum and on the inner and outer membranes of the nuclear envelope , requiring appropriate membrane permeabilization.

  • Overfixation can mask epitopes while insufficient fixation may lead to protein redistribution artifacts.

  • A comparison of different fixation methods (paraformaldehyde, methanol, acetone) is recommended to identify optimal conditions.

• Resolution and detection method:

  • Confocal microscopy provides better resolution for subcellular localization studies compared to conventional fluorescence microscopy.

  • Super-resolution microscopy techniques can offer even more precise localization information.

  • Subcellular fractionation followed by immunoblotting can complement imaging approaches.

• Controls for subcellular localization:

  • Co-staining with established markers for subcellular compartments is essential.

  • Validation with multiple antibodies recognizing different epitopes can increase confidence.

  • Correlation with functional studies (e.g., enzyme activity assays in isolated subcellular fractions).

When studying subcellular localization changes during cellular activation, it's important to note that changes in COX2 distribution might be accompanied by changes in expression level. Time-course experiments with careful quantification are necessary to distinguish between these phenomena.

How can I troubleshoot non-specific binding and background issues when using COX2 antibodies?

Non-specific binding and high background are common challenges when working with COX2 antibodies. Here are systematic approaches to troubleshoot these issues:

For Western blotting:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, commercial blockers) and concentrations.

  • Extend blocking time if background remains high.

• Adjust antibody conditions:

  • Titrate primary antibody concentration to find optimal signal-to-noise ratio.

  • Increase washing duration and number of washes between antibody incubations.

  • Consider overnight incubation at 4°C rather than shorter incubations at room temperature.

• Sample preparation:

  • Ensure complete lysis and denaturation of samples.

  • Consider including reducing agents if the antibody recognizes a linear epitope.

  • Filter lysates to remove particulates that may cause spotting.

For immunohistochemistry/immunofluorescence:

• Tissue/cell preparation:

  • Optimize fixation time to prevent overfixation.

  • Include peroxidase blocking step for tissues with high endogenous peroxidase.

  • Block endogenous biotin if using biotinylated secondary antibodies.

• Antibody optimization:

  • Perform antibody titration experiments.

  • Consider using antibody diluents containing background reducers.

  • Extend washing steps between antibody incubations.

• Controls to include:

  • No primary antibody control to assess secondary antibody background.

  • Isotype control to evaluate non-specific binding.

  • Pre-absorption with immunizing peptide to confirm specificity.

  • Tissues known to be negative for COX2 (under non-inflammatory conditions) as negative controls.

If cross-reactivity with COX1 is suspected, validation experiments should include samples with differential expression of COX1 and COX2 (e.g., tissues from COX2-knockout models or cells where COX2 has been silenced).

What are the best practices for quantifying COX2 expression levels in heterogeneous tissue samples?

Quantifying COX2 expression in heterogeneous tissues requires careful methodological consideration:

• Immunohistochemical approach:

  • Use validated antibodies with established specificity .

  • Employ digital image analysis software for unbiased quantification.

  • Establish scoring systems that account for both staining intensity and percentage of positive cells.

  • Consider using tissue microarrays for high-throughput analysis.

• Cell type-specific analysis:

  • Implement multiplex immunofluorescence to simultaneously detect COX2 and cell-type markers.

  • Consider single-cell approaches such as flow cytometry for tissues that can be dissociated.

  • Laser capture microdissection can isolate specific cell populations for subsequent analysis.

• Normalization strategies:

  • Include internal controls (housekeeping proteins) for Western blot quantification.

  • For immunohistochemistry, normalize to tissue area or cell count.

  • Consider batch effects when comparing samples processed at different times.

• Statistical considerations:

  • Account for intra-tumoral heterogeneity by sampling multiple regions.

  • Apply appropriate statistical tests for non-normal distributions often found in expression data.

  • Consider the biological relevance of threshold values when categorizing expression levels.

• Validation approaches:

  • Correlate protein expression with mRNA levels (qPCR or RNA-seq).

  • Validate findings with multiple antibodies or alternative detection methods.

  • Include functional assays to correlate expression with enzymatic activity.

When reporting COX2 expression data, it's important to clearly describe the quantification methodology, including antibody details, scoring system, and normalization approach to facilitate comparison across studies.

What controls should be included to validate COX2 antibody specificity in different experimental applications?

Proper controls are essential for validating COX2 antibody specificity across different applications:

For Western blotting:

• Positive controls:

  • Lysates from cells known to express COX2 (e.g., macrophages stimulated with LPS or IL-1α) .

  • Recombinant COX2 protein as a size reference.

• Negative controls:

  • Lysates from COX2 knockout cells/tissues.

  • Samples from cells treated with COX2-specific siRNA.

• Specificity controls:

  • Pre-incubation of antibody with immunizing peptide to confirm binding specificity.

  • Comparison with multiple antibodies recognizing different COX2 epitopes.

  • Parallel blotting with COX1 antibodies to confirm isoform specificity.

For immunohistochemistry/immunofluorescence:

• Positive controls:

  • Tissues with known COX2 expression (e.g., inflamed tissues).

  • Cell lines with confirmed COX2 expression.

• Negative controls:

  • Primary antibody omission to assess secondary antibody background.

  • Isotype control antibodies to evaluate non-specific binding.

  • Tissues from COX2 knockout models when available.

• Specificity controls:

  • Peptide competition assays.

  • Correlation with in situ hybridization for COX2 mRNA.

  • Comparison of staining patterns across multiple antibodies.

For immunoprecipitation:

• Input control to confirm target protein presence in starting material.

• Non-specific IgG control to assess background binding.

• Reverse IP using antibodies against suspected interacting proteins.

Additional validation approaches:

• Antibody validation in cells with manipulated COX2 expression (overexpression or knockdown).

• Correlation of antibody signal with functional assays of COX2 activity.

• Mass spectrometry confirmation of immunoprecipitated proteins.

Proper documentation of these controls is essential for publishing results and ensuring reproducibility across laboratories.

How should researchers interpret discrepancies between COX2 protein levels and enzymatic activity in experimental models?

Discrepancies between COX2 protein levels (detected by antibodies) and enzymatic activity are common and can provide valuable insights into COX2 regulation. Researchers should consider several factors when interpreting such discrepancies:

• Posttranslational modifications:

  • Recent studies have observed that the kinetics of prostaglandin synthesis doesn't always correlate with COX2 protein expression, suggesting posttranslational regulation .

  • COX2 enzymatic activity can be regulated by s-nitrosylation, glycosylation, and phosphorylation without changes in protein level .

  • These modifications might not affect antibody detection but can significantly impact enzyme function.

• Protein-protein interactions:

  • Proteins like Fyn and ELMO1 can modulate COX2 enzymatic activity without altering protein levels .

  • These interactions might affect COX2 activity either through conformational changes or by impacting subcellular localization .

  • Consider assessing known COX2 interacting partners when activity doesn't correspond with expression.

• Substrate availability:

  • COX2 activity depends on the availability of arachidonic acid, which is regulated by phospholipase A2 activity .

  • Measuring substrate levels alongside COX2 protein can help explain activity discrepancies.

• Subcellular localization:

  • COX2 is located on the lumenal surface of the endoplasmic reticulum and nuclear envelope membranes .

  • Changes in localization might affect access to substrate or interaction with regulatory proteins.

• Methodological considerations:

  • Ensure that activity assays are specific for COX2 (vs. COX1) by using selective inhibitors.

  • Consider the stability of prostaglandins being measured as endpoints of COX2 activity.

  • Time-course experiments may reveal temporal disconnects between protein expression and activity.

When publishing research involving COX2, it's valuable to include both protein expression and activity measurements, along with assessment of potential regulatory mechanisms when discrepancies are observed.

What are the considerations for comparative analysis of COX2 expression across different cell types and disease models?

Comparative analysis of COX2 expression across different experimental systems requires careful consideration of multiple factors:

• Baseline expression differences:

  • COX2 is constitutively expressed in some tissues (endothelium, kidney, brain) but not in others .

  • Establish appropriate baseline controls specific to each cell type or tissue.

• Induction kinetics:

  • The timing of COX2 upregulation after stimulation varies across cell types.

  • Design time-course experiments to capture peak expression in each system.

  • Consider that different inducers (cytokines, growth factors, stress) may have cell-type specific effects on COX2 induction .

• Methodological standardization:

  • Use the same antibody clone and detection protocol across all samples for direct comparison.

  • Standardize protein loading controls and quantification methods.

  • Include calibration standards when possible (e.g., recombinant protein).

• Reporting standards:

  • Clearly specify cell types, disease models, and experimental conditions.

  • Report fold-changes relative to appropriate controls rather than absolute values.

  • Include statistical analysis appropriate for the data distribution.

• Functional correlation:

  • Correlate expression data with functional outcomes (e.g., prostaglandin production).

  • Consider that the same level of COX2 expression might have different functional impacts in different cell types.

• Multi-omics integration:

  • Integrate protein expression data with transcriptomic data to identify potential post-transcriptional regulation.

  • Consider epigenetic factors that might influence cell-type specific expression patterns.

• Disease context interpretation:

  • COX2 overexpression has been reported in various diseases including cancer and inflammatory conditions .

  • The pathophysiological significance of COX2 expression may differ between disease models.

  • Consider treatment effects on COX2 expression when studying therapeutic interventions.

When publishing comparative studies, provide detailed methodological information and consider presenting normalized data alongside raw data to facilitate cross-study comparisons.

How can researchers distinguish between COX2 expression changes due to transcriptional regulation versus post-transcriptional mechanisms?

Distinguishing between different regulatory mechanisms affecting COX2 expression requires a multi-faceted experimental approach:

• Comparative mRNA and protein analysis:

  • Measure COX2 mRNA (by qRT-PCR or RNA-seq) and protein (by Western blot or immunohistochemistry) in parallel.

  • Discordance between mRNA and protein levels suggests post-transcriptional regulation.

  • Time-course experiments can reveal temporal relationships between mRNA and protein changes.

• Transcriptional regulation assessment:

  • Analyze promoter activity using reporter assays with COX2 promoter constructs.

  • Examine binding of transcription factors to the COX2 promoter using ChIP assays.

  • Assess epigenetic modifications (DNA methylation, histone modifications) at the COX2 locus.

• Post-transcriptional regulation analysis:

  • Measure COX2 mRNA stability using actinomycin D chase experiments or similar approaches.

  • Investigate potential microRNA regulation using prediction algorithms and validation experiments.

  • Assess polysome association of COX2 mRNA to evaluate translational efficiency.

• Post-translational regulation investigation:

  • Examine protein stability using cycloheximide chase experiments.

  • Investigate ubiquitination status and proteasomal degradation.

  • COX2 has been shown to be regulated by accelerated degradation via the proteasome in some contexts .

• Specific regulatory mechanisms to consider:

  • GPCRs have been shown to downregulate COX2 by accelerating its degradation via the proteasome without affecting protein synthesis .

  • Glucocorticoids chronically trans-repress the PTGS2 gene by interfering with transcription initiation and elongation .

  • Specific proteins may interact with COX2 to modulate its stability or activity without affecting transcription .

• Experimental interventions:

  • Use proteasome inhibitors to assess contribution of protein degradation.

  • Apply transcription inhibitors to distinguish between transcriptional and post-transcriptional events.

  • Employ translation inhibitors to identify regulation at the translational level.

By systematically addressing these aspects, researchers can build a comprehensive understanding of the regulatory mechanisms controlling COX2 expression in their experimental system.

What are the promising approaches for studying COX2 in single-cell analyses and spatial transcriptomics?

Emerging technologies offer new opportunities for studying COX2 at single-cell resolution and in spatial contexts:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) can detect COX2 protein alongside dozens of other markers in single cells.

  • The AS67 monoclonal antibody has been validated for flow cytometry applications, which can be adapted for single-cell analysis .

  • Imaging mass cytometry combines antibody-based detection with spatial resolution at subcellular levels.

• Spatial transcriptomics integration:

  • In situ hybridization techniques like RNAscope can visualize COX2 mRNA within tissue architecture.

  • Combined immunofluorescence for COX2 protein and RNA-FISH for COX2 mRNA can reveal spatial discordances between transcription and translation.

  • Spatial proteomics approaches using multiplexed antibody staining can map COX2 expression within tissue microenvironments.

• Single-cell multi-omics approaches:

  • CITE-seq can simultaneously profile COX2 protein (using antibodies) and transcriptome in single cells.

  • REAP-seq offers similar capabilities with potentially higher sensitivity for protein detection.

  • These approaches can reveal heterogeneity in COX2 expression and regulation within seemingly homogeneous populations.

• Live-cell imaging applications:

  • Antibody fragments (Fab) conjugated to fluorophores can track COX2 in living cells.

  • CRISPR-based tagging of endogenous COX2 with fluorescent proteins allows long-term tracking.

  • These approaches can provide insights into dynamics of COX2 expression and localization.

• Methodological considerations:

  • Validation of antibody specificity is particularly crucial in multiplexed and single-cell applications.

  • Cell fixation and permeabilization protocols may need optimization for these specialized techniques.

  • Computational analysis pipelines should account for technical variables in single-cell data.

• Emerging applications:

  • Single-cell analysis of COX2 in tumor microenvironments can reveal cellular subpopulations driving inflammation.

  • Spatial mapping of COX2 at tissue interfaces (e.g., tumor-stroma) can provide insights into intercellular communication.

  • Temporal single-cell analysis during inflammatory responses can capture the dynamics of COX2 regulation.

These approaches offer unprecedented resolution for studying COX2 biology but require careful technical validation and integration with functional assays to establish biological significance.

How can researchers effectively combine COX2 antibody-based detection with functional activity assays in complex experimental systems?

Integrating structural detection with functional assessment provides a more comprehensive understanding of COX2 biology. Here are effective approaches:

• Sequential analysis from the same sample:

  • Split samples for parallel protein detection and activity assays.

  • Use adjacent tissue sections for immunohistochemistry and ex vivo activity measurements.

  • Develop protocols that allow extraction of both protein (for immunoblotting) and metabolites (for activity assessment) from the same specimen.

• Complementary functional assays:

  • Measure prostaglandin production (PGE2, PGD2, etc.) by ELISA or mass spectrometry.

  • Utilize selective COX2 inhibitors to confirm specificity of detected prostaglandin production.

  • Consider measuring multiple prostanoids to capture the full spectrum of COX2 activity.

• In situ activity detection:

  • Combine immunofluorescence with fluorescent activity-based probes when available.

  • Use proximity ligation assays to detect COX2 interaction with substrate or regulatory proteins.

  • Consider reporter cell lines expressing fluorescent or luminescent indicators of COX2 pathway activation.

• Temporal coordination:

  • Design time-course experiments capturing both protein expression and activity dynamics.

  • Account for potential time lags between protein expression and detectable enzymatic activity.

  • Include both early and late time points to capture transient versus sustained responses.

• Technical considerations:

  • Standardize experimental conditions affecting enzyme activity (temperature, pH, oxygen tension).

  • Include appropriate controls for both antibody specificity and activity assay selectivity.

  • Consider that sample processing for antibody detection might affect functional measurements.

• Data integration approaches:

  • Develop correlation analyses between protein levels and activity measurements.

  • Consider mathematical modeling to account for non-linear relationships.

  • Use multivariate statistical approaches when integrating multiple parameters.

• Advanced systems:

  • In organoid cultures, combine immunostaining with media collection for secreted prostanoids.

  • For in vivo studies, correlate tissue immunohistochemistry with biofluid prostaglandin measurements.

  • In patient-derived samples, relate COX2 immunohistochemistry scores with clinical response to COX2 inhibitors.

This integrated approach can reveal important insights about posttranslational regulation and identify situations where COX2 protein expression does not correlate with enzymatic activity due to regulatory mechanisms .

What are the emerging approaches for studying COX2 protein-protein interactions that regulate its function?

Recent advances have expanded the toolkit for studying COX2 protein-protein interactions, offering new insights into its regulatory mechanisms:

• Proximity-based labeling techniques:

  • BioID or TurboID fusion constructs with COX2 can identify proteins in close proximity in living cells.

  • APEX2 fusion proteins provide higher temporal resolution for capturing dynamic interactions.

  • These approaches can identify novel interaction partners that may be missed by traditional co-immunoprecipitation due to weak or transient interactions.

• Advanced imaging approaches:

  • FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) can visualize direct protein interactions in living cells.

  • Super-resolution microscopy combined with dual immunolabeling can visualize co-localization at nanoscale resolution.

  • FLIM (Fluorescence Lifetime Imaging Microscopy) can detect protein interactions with minimal perturbation of cellular systems.

• MS-based interactomics:

  • Quantitative proteomics following COX2 immunoprecipitation can identify stimulus-dependent changes in the COX2 interactome.

  • Crosslinking mass spectrometry (XL-MS) can map interaction interfaces at amino acid resolution.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify conformational changes upon protein binding.

• Functional validation strategies:

  • Domain mapping through truncation or point mutations to identify interaction interfaces.

  • Peptide competition assays to disrupt specific interactions.

  • Genetic approaches (CRISPR/Cas9) to modify endogenous proteins and validate interactions in physiological contexts.

• Specific protein interactions of interest:

  • Recent data identified proteins located in close proximity to COX2 enzyme that serve as posttranslational modulators of COX2 function, upregulating its enzymatic activity .

  • Proteins like Fyn and ELMO1 have been identified as regulatory partners of COX2 .

  • These interactions may regulate COX2 activity through conformational changes or by affecting subcellular localization .

• Computational approaches:

  • Molecular docking and molecular dynamics simulations can predict and characterize interaction interfaces.

  • Network analysis of proteomics data can identify hub proteins in COX2 regulatory networks.

  • Machine learning approaches can predict functional consequences of specific interactions.

When designing studies of COX2 protein interactions, researchers should consider that these interactions may be tissue-specific, stimulus-dependent, or influenced by posttranslational modifications of COX2 itself. The integration of multiple complementary techniques provides the most robust characterization of COX2 protein interaction networks.