IRF3 Antibody

What is IRF3 and why is it significant in immunological research?

IRF3 is a key transcriptional regulator of type I interferon (IFN)-dependent immune responses that plays a critical role in the innate immune response against DNA and RNA viruses. It functions as a ~55-60 kDa constitutively expressed member of the IRF family . IRF3 regulates the transcription of type I IFN genes (IFN-alpha and IFN-beta) and IFN-stimulated genes (ISG) by binding to an interferon-stimulated response element (ISRE) in their promoters . It acts as a more potent activator of the IFN-beta gene than the IFN-alpha gene and plays a critical role in both early and late phases of IFN gene induction . Its significance extends to SARS-CoV-2 infection response, making it a crucial target for immunological and virological research .

What structural and functional domains characterize IRF3 protein?

IRF3 contains several functional domains that determine its activity and regulation:

• DNA binding domain (aa 7-107)

• Nuclear export signal (aa 139-149)

• Multiple phosphorylation sites (aa 395-407)

• C-terminal regulatory domain (targeted by antibodies like D-3 between aa 389-427)

The protein exists in an inactive form in the cytoplasm of uninfected cells. Following viral infection, double-stranded RNA (dsRNA), or toll-like receptor (TLR) signaling, IRF3 becomes phosphorylated by IKBKE and TBK1 kinases . This phosphorylation induces a conformational change, leading to dimerization and nuclear translocation, where it associates with CREB binding protein (CREBBP) to form dsRNA-activated factor 1 (DRAF1) . This complex then activates the transcription of type I IFN and ISG genes, orchestrating the antiviral response .

What applications are IRF3 antibodies most commonly validated for?

IRF3 antibodies have been validated for multiple research applications:

Selection should be based on the specific experimental requirements and the validated applications of each antibody clone .

How should I select the appropriate IRF3 antibody for my specific research application?

When selecting an IRF3 antibody, consider these key factors:

• Antibody Format: Choose between:

  • Monoclonal (like clone D-3, 3F10, 482205, IRF35I218) for high specificity

  • Polyclonal (like DF6895) for broader epitope recognition

  • Recombinant multiclonal for consistent lot-to-lot performance

• Target Species: Verify cross-reactivity with your experimental model:

  • Human IRF3 (all antibodies in search results)

  • Mouse/rat IRF3 (select antibodies like D-3)

• Application Compatibility: Ensure validation for your technique:

  • For Western blotting: Most antibodies are validated

  • For immunofluorescence/immunohistochemistry: Check specific validation

  • For flow cytometry: Consider conjugated antibodies like Alexa Fluor 488-conjugated antibodies

• Epitope Location: Select based on your research question:

  • N-terminal antibodies (aa 1-150) for DNA binding domain studies

  • C-terminal antibodies (aa 389-427) for phosphorylation studies

  • Mid-region antibodies (aa 108-166, 206-427) for specific applications

• Conjugation Requirements: Choose between:

  • Unconjugated for flexibility in detection methods

  • Directly conjugated (Alexa Fluor 488, HRP, PE, FITC) for direct detection

Match the antibody specifications to your experimental design for optimal results .

What controls should I incorporate when using IRF3 antibodies in experimental workflows?

Implementing proper controls is essential for reliable IRF3 antibody experiments:

• Positive Controls:

  • Cell lines known to express IRF3 (e.g., HeLa, Daudi)

  • Recombinant IRF3 protein for western blotting

  • Cells treated with poly(I:C) or viral stimuli to induce IRF3 activation

• Negative Controls:

  • Isotype-matched control antibodies (e.g., mouse IgG1 for D-3, mouse IgG2B for 482205)

  • IRF3 knockout or knockdown cells/tissues

  • Blocking peptide competition to confirm specificity

• Phosphorylation-State Controls:

  • Unstimulated cells (inactive IRF3)

  • Phosphatase-treated samples to eliminate phosphorylated forms

• Subcellular Localization Controls:

  • Nuclear/cytoplasmic fractionation markers

  • Time-course of stimulation to capture translocation dynamics

• Application-Specific Controls:

  • For flow cytometry: Unstained cells and permeabilization controls

  • For immunoprecipitation: Non-specific IgG precipitation control

  • For western blotting: Loading controls (β-actin, GAPDH)

Incorporating these controls ensures specificity, validates responses, and allows accurate interpretation of experimental results .

How can I distinguish between inactive and phosphorylated (active) forms of IRF3?

Differentiating between inactive and phosphorylated IRF3 requires specific techniques:

• Phospho-specific antibodies: Though not mentioned in the search results, commercial phospho-specific antibodies that recognize Ser396, Ser386, and other phosphorylation sites are available and essential for direct detection of activated IRF3.

• Mobility shift detection: Phosphorylated IRF3 migrates more slowly in SDS-PAGE, appearing as higher molecular weight bands compared to inactive IRF3 . Use lower percentage gels (7-8%) and longer running times to improve separation.

• Nuclear/cytoplasmic fractionation: Since phosphorylated IRF3 translocates to the nucleus, comparing nuclear vs. cytoplasmic fractions can indirectly indicate activation state . Use subcellular fractionation followed by western blotting with IRF3 antibodies.

• Dimerization analysis: Activated IRF3 forms dimers. Use non-denaturing gel electrophoresis or chemical crosslinking followed by SDS-PAGE to detect dimeric forms .

• Immunofluorescence microscopy: Monitor IRF3 nuclear translocation as a proxy for activation. Inactive IRF3 appears predominantly cytoplasmic, while activated IRF3 shows nuclear accumulation .

• Co-immunoprecipitation with CBP/p300: Active IRF3 associates with CREB binding protein to form DRAF1 complex. Co-IP experiments can detect this interaction using antibodies like D-3 that work in IP applications .

Combining these approaches provides comprehensive analysis of IRF3 activation status in experimental systems .

What are optimal protocols for detecting IRF3 by western blotting?

For successful IRF3 western blotting, follow these optimized protocols:

• Sample Preparation:

  • Lyse cells in RIPA or NP-40 buffer with protease and phosphatase inhibitors

  • For detecting phosphorylated forms, add phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Use fresh samples when possible or snap-freeze and store at -80°C

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels for standard detection

  • Consider 7-8% gels for better separation of phosphorylated forms

  • Load 20-50 μg total protein per lane for cell lysates

• Transfer and Blocking:

  • Transfer to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST (use BSA for phospho-detection)

  • For cleaner results with specific antibodies like 3F10, use optimized blocking solutions

• Antibody Incubation:

  • Primary antibody dilutions:

    • D-3 (sc-376455): Use at 1:100-1:1000 dilution

    • Clone 3F10: Use at 1:1000-1:3000 dilution

    • Other clones: Follow manufacturer recommendations

  • Incubate overnight at 4°C for optimal results

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000

• Detection:

  • Use ECL or more sensitive chemiluminescent substrates for detection

  • Expected band size: ~55-60 kDa for full-length IRF3

  • Note that phosphorylated forms may appear as higher molecular weight bands

• Troubleshooting Tips:

  • Multiple bands may indicate phosphorylation states, splice variants, or degradation products

  • High background: Increase washing steps or adjust antibody concentration

  • No signal: Verify protein expression, adjust lysate concentration, check transfer efficiency

Following these protocols will produce reliable western blot results for IRF3 detection .

What are the best approaches for studying IRF3 nuclear translocation?

Studying IRF3 nuclear translocation effectively requires these methodological approaches:

• Immunofluorescence Microscopy:

  • Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.5% Triton X-100 (5-10 minutes)

  • Block with 1-5% BSA or normal serum

  • Incubate with IRF3 antibodies validated for IF applications (e.g., D-3, IRF35I218)

  • Co-stain with DAPI for nuclear visualization

  • Use confocal microscopy for highest resolution of nuclear/cytoplasmic distinction

• Subcellular Fractionation:

  • Separate nuclear and cytoplasmic fractions using commercial kits or established protocols

  • Analyze fractions by western blotting using antibodies like D-3, 3F10, or IRF35I218

  • Include markers for nuclear (e.g., lamin, histone) and cytoplasmic (e.g., GAPDH, tubulin) fractions

  • Quantify the nuclear/cytoplasmic ratio of IRF3

• Time-Course Experiments:

  • Stimulate cells with appropriate triggers (viral infection, poly(I:C), LPS)

  • Collect samples at multiple timepoints (0, 30, 60, 90, 120, 240 minutes)

  • Process for IF or fractionation to capture the dynamics of translocation

  • Consider phospho-IRF3 antibodies to correlate phosphorylation with translocation

• Live-Cell Imaging:

  • Create IRF3-fluorescent protein fusions (e.g., GFP-IRF3)

  • Perform time-lapse imaging after stimulation

  • Quantify nuclear accumulation over time

• Flow Cytometry:

  • Use Alexa Fluor 488-conjugated IRF3 antibodies after proper fixation and permeabilization

  • Compare nuclear staining intensity pre- and post-stimulation

  • Follow protocols like those used for the Daudi cell line in search result #3

• Image Analysis:

  • Use software to quantify nuclear/cytoplasmic fluorescence intensity ratios

  • Analyze at least 50-100 cells per condition for statistical significance

  • Plot translocation kinetics to determine rate and extent of nuclear accumulation

These approaches can be used individually or in combination for comprehensive analysis of IRF3 nuclear translocation dynamics .

How can IRF3 antibodies be utilized to study virus-host interactions?

IRF3 antibodies are valuable tools for investigating virus-host interactions:

• Viral Evasion Mechanisms:

  • Use western blotting and immunoprecipitation to detect virus-induced IRF3 degradation or modification

  • Compare IRF3 phosphorylation levels between mock-infected and virus-infected cells

  • Investigate viral proteins that directly interact with IRF3 using co-immunoprecipitation with antibodies like D-3

• Type I Interferon Response Dynamics:

  • Monitor IRF3 nuclear translocation kinetics during infection using immunofluorescence

  • Correlate IRF3 activation with interferon production using paired samples

  • Study how different viral strains affect IRF3 activation patterns

• SARS-CoV-2 Research:

  • Investigate IRF3 activation during SARS-CoV-2 infection, as IRF3 is a key transcription factor in this context

  • Compare wild-type virus with variants to determine effects on IRF3 signaling

  • Study interactions between viral proteins and IRF3 pathway components

• Cell-Type Specific Responses:

  • Compare IRF3 activation patterns across different cell types (epithelial cells, macrophages, dendritic cells)

  • Use flow cytometry with Alexa Fluor 488-conjugated IRF3 antibodies for cell-type specific analysis

  • Investigate tissue-specific IRF3 activation in infected animal models using IHC

• Apoptosis Pathway Investigation:

  • Study the formation of the apoptosis complex (TOMM70:HSP90AA1:IRF3:BAX) during Sendai virus infection

  • Use co-immunoprecipitation with IRF3 antibodies to pull down complex components

  • Correlate IRF3 activation with apoptotic markers in infected cells

• Therapeutic Intervention Assessment:

  • Evaluate how antivirals or immunomodulators affect IRF3 activation during infection

  • Screen for compounds that enhance or inhibit IRF3 signaling as potential therapeutic approaches

  • Use phospho-IRF3 detection to measure pathway modulation

These applications demonstrate how IRF3 antibodies serve as critical reagents for understanding virus-host interactions and developing interventions for viral diseases .

What methods can be employed to study IRF3-mediated gene regulation?

Investigating IRF3-mediated gene regulation requires specialized techniques:

• Chromatin Immunoprecipitation (ChIP):

  • Use IRF3 antibodies to immunoprecipitate chromatin-bound IRF3

  • Detect binding to interferon-stimulated response elements (ISREs) in target promoters

  • Select antibodies validated for IP applications like D-3

  • Follow with qPCR or sequencing (ChIP-seq) to identify binding sites genome-wide

• Electrophoretic Mobility Shift Assay (EMSA):

  • Detect IRF3 binding to DNA probes containing ISRE motifs

  • Use IRF3 antibodies for supershift assays to confirm specificity

  • Compare nuclear extracts from stimulated vs. unstimulated cells

• Reporter Gene Assays:

  • Construct luciferase reporters driven by IRF3-responsive promoters (IFN-β, ISGs)

  • Co-transfect with wild-type or mutant IRF3 constructs

  • Use IRF3 antibodies to confirm expression levels by western blotting

• Co-Immunoprecipitation with Transcriptional Co-factors:

  • Use IRF3 antibodies to pull down complexes containing CREB binding protein (CBP/p300)

  • Investigate formation of dsRNA-activated factor 1 (DRAF1)

  • Identify additional interacting partners in the transcriptional complex

• RNA Analysis:

  • Perform RT-qPCR to measure expression of IRF3 target genes after stimulation

  • Compare with IRF3 activation status detected by western blotting

  • Consider RNA-seq for genome-wide analysis of IRF3-dependent gene expression

• Proteomics Approaches:

  • Use mass spectrometry to identify IRF3-interacting proteins after immunoprecipitation

  • Study post-translational modifications of IRF3 that affect its transcriptional activity

  • Compare protein interaction networks in different activation states

• CRISPR/Cas9 Gene Editing:

  • Generate IRF3 knockout or mutant cell lines

  • Use IRF3 antibodies to confirm knockout efficiency

  • Compare transcriptional responses between wild-type and modified cells

These methodologies provide comprehensive insights into the mechanisms and targets of IRF3-mediated gene regulation in diverse biological contexts .

How should I interpret multiple bands in IRF3 western blots?

Multiple bands in IRF3 western blots can provide valuable biological information when properly interpreted:

• Phosphorylation States:

  • Higher molecular weight bands (above the main 55-60 kDa band) often represent phosphorylated forms

  • Increasing intensity of these upper bands after stimulation indicates IRF3 activation

  • The pattern of multiple closely spaced higher bands may reflect different phosphorylation combinations on serine/threonine residues

• Splice Variants:

  • Search results indicate several possible splice variants of IRF3:

    • Deletion of aa 201-327

    • Alternative start site at Met147

    • 125 aa substitution for C-terminal 100 aa (aa 328-427)

  • These variants may appear as lower molecular weight bands

• Degradation Products:

  • Bands below the main IRF3 band might represent proteolytic fragments

  • Increasing lower bands during viral infection could indicate viral-induced degradation

  • Include protease inhibitors in lysis buffers to minimize artifactual degradation

• Antibody-Specific Patterns:

  • Different antibody clones targeting distinct epitopes may recognize different forms

  • C-terminal antibodies like D-3 (aa 389-427) would not detect C-terminal truncated variants

  • N-terminal antibodies like IRF35I218 (aa 1-150) would not detect N-terminal truncated forms

• Analytical Approach:

  • Use phosphatase treatment controls to identify phosphorylation-dependent bands

  • Compare patterns across different cell types and stimulation conditions

  • Consider using multiple antibodies recognizing different epitopes

  • For critical experiments, confirm with knockout/knockdown controls

• Common Band Patterns:

  • ~55-60 kDa: Full-length IRF3

  • ~70-75 kDa: Hyperphosphorylated IRF3 after stimulation

  • ~40-45 kDa: Potential splice variants or degradation products

  • ~100-110 kDa: Potential dimers or IRF3 complexed with other proteins

Understanding these patterns enables accurate interpretation of IRF3 biology in experimental systems .

What are the key considerations when quantifying IRF3 activation?

Quantifying IRF3 activation requires careful attention to several methodological considerations:

• Western Blot Quantification:

  • Calculate the ratio of phosphorylated to total IRF3

  • Use densitometry software to measure band intensities

  • Normalize to appropriate loading controls (β-actin, GAPDH)

  • Present data as fold-change relative to unstimulated conditions

  • Consider the dynamic range limitations of western blotting

• Nuclear Translocation Measurement:

  • In immunofluorescence: Calculate nuclear/cytoplasmic intensity ratios

  • In subcellular fractionation: Measure IRF3 band intensity in nuclear vs. cytoplasmic fractions

  • Account for background signal and normalize to fraction-specific markers

  • Analyze sufficient cell numbers (>50-100) for statistical significance

• Functional Readouts:

  • Measure downstream gene expression (IFN-β, ISGs) as proxy for IRF3 activity

  • Correlate with direct IRF3 measurements for comprehensive assessment

  • Consider luciferase reporter assays with ISRE-containing promoters

• Time-Course Considerations:

  • IRF3 activation is dynamic with distinct phases

  • Early activation (30-60 minutes): Initial phosphorylation

  • Peak activation (1-4 hours): Maximum nuclear translocation and target gene induction

  • Resolution phase: Dephosphorylation or degradation

  • Design sampling timepoints accordingly

• Statistical Analysis:

  • Perform experiments in biological triplicates minimum

  • Use appropriate statistical tests (t-test, ANOVA) with post-hoc corrections

  • Report both statistical significance and effect size

  • Consider variability between cell types and stimulation conditions

• Validation Approaches:

  • Verify with multiple detection methods (western blot, IF, reporter assays)

  • Include positive controls (known IRF3 activators like poly(I:C))

  • Use IRF3 inhibitors or knockdown as negative controls

  • Consider alternative measurement techniques for confirmation

These considerations ensure robust and reproducible quantification of IRF3 activation across different experimental paradigms .

How can IRF3 antibodies contribute to SARS-CoV-2 and emerging virus research?

IRF3 antibodies provide valuable tools for investigating SARS-CoV-2 and emerging viral pathogenesis:

• Virus-Host Interaction Mechanisms:

  • Investigate how SARS-CoV-2 proteins interact with and potentially antagonize IRF3 signaling

  • Compare IRF3 activation patterns between different coronavirus variants

  • Study kinetics of IRF3 activation during infection using time-course experiments with IRF3 antibodies

• Cell-Type Specific Responses:

  • Examine IRF3 activation in different respiratory cell types (epithelial cells, alveolar macrophages)

  • Use immunohistochemistry with IRF3 antibodies on COVID-19 patient samples

  • Compare with other respiratory viruses to identify unique signatures

• Therapeutic Development:

  • Screen compounds that modulate IRF3 signaling as potential antivirals

  • Use IRF3 antibodies to monitor pathway activation in drug screening assays

  • Evaluate how repurposed drugs affect IRF3-dependent antiviral responses

• Vaccine Research:

  • Study how vaccine adjuvants activate IRF3 signaling

  • Compare innate immune signatures between different vaccine platforms

  • Use IRF3 antibodies to characterize the initial immune response to vaccination

• Long COVID Investigations:

  • Examine persistent alterations in IRF3 signaling in long COVID patients

  • Compare tissue samples from acute vs. long COVID cases using IHC

  • Investigate potential autoimmune aspects involving IRF3 pathway dysregulation

• Emerging Pathogen Preparedness:

  • Develop standardized IRF3 activation assays for rapid evaluation of novel pathogens

  • Create biosensor cell lines with IRF3-dependent reporters for pathogen screening

  • Establish IRF3 antibody-based diagnostic approaches for viral infections

IRF3 antibodies thus serve as critical reagents for understanding the pathogenesis of SARS-CoV-2 and other emerging viruses, with direct implications for therapeutic and preventive strategies .

What is the role of IRF3 in non-viral pathological conditions and how can antibodies help investigate this?

Beyond viral infections, IRF3 plays important roles in various physiological and pathological processes:

• Autoimmune Diseases:

  • IRF3 contributes to inflammatory pathways in autoimmune conditions

  • Use IRF3 antibodies to assess activation status in patient samples

  • Compare phosphorylation patterns and nuclear localization in affected tissues

  • Investigate potential dysregulation in type I interferon-mediated pathologies

• Cancer Biology:

  • IRF3 can influence apoptotic pathways and potentially affect tumor development

  • The apoptosis complex (TOMM70:HSP90AA1:IRF3:BAX) identified in search results suggests cancer relevance

  • Use immunohistochemistry with IRF3 antibodies to evaluate expression in tumor tissues

  • Investigate correlations between IRF3 activation status and cancer progression

• Sterile Inflammation:

  • IRF3 responds to damage-associated molecular patterns (DAMPs) in non-infectious inflammation

  • Study IRF3 activation in models of ischemia-reperfusion injury or trauma

  • Use western blotting and immunofluorescence to track IRF3 in inflammatory conditions

• Metabolic Disorders:

  • Emerging evidence suggests IRF3 involvement in metabolic regulation

  • Investigate IRF3 activation in adipose tissue, liver, and muscle in metabolic disease models

  • Use IRF3 antibodies to study tissue-specific activation patterns

• Neurological Conditions:

  • IRF3 signaling occurs in microglia and affects neuroinflammation

  • Examine IRF3 activation in neurodegenerative disease models

  • Use immunofluorescence with IRF3 antibodies to study microglial activation

• Research Methodologies:

  • Immunoprecipitation to identify novel IRF3 interaction partners in different disease contexts

  • ChIP-seq to map IRF3 binding sites in different cell types and pathological states

  • Tissue microarrays with IRF3 staining to screen multiple patient samples

These applications demonstrate how IRF3 antibodies facilitate investigation of diverse pathological processes beyond viral infections, potentially identifying new therapeutic targets and biomarkers .