STAT2 Antibody

What is STAT2 and why is it important in research?

STAT2 (Signal Transducer and Activator of Transcription 2) is a critical transcription factor in the type I interferon (IFN) signaling pathway. It functions primarily as a heterodimer with STAT1, associating with IRF9 to form the ISGF3 transcription factor complex. STAT2 mediates signaling by type I IFNs (IFN-alpha and IFN-beta) following their binding to cell surface receptors. This triggers the activation of Janus kinases (TYK2 and JAK1), leading to tyrosine phosphorylation of STAT2 and STAT1 .

STAT2 is particularly important in research because:

• It's essential for antiviral immunity and protection against viral infections

• It regulates the expression of interferon-stimulated genes (ISGs)

• It has been implicated in tumor immunity and cancer biology

• STAT2 deficiency has been linked to increased susceptibility to viral infections in humans

What are the known structural characteristics of human STAT2 protein?

Human STAT2 is an 851 amino acid protein with several key structural domains:

The protein contains regions that are important for DNA binding, transcriptional activation, and interaction with other proteins, including JAK kinases and STAT1 .

How can I verify the specificity of a STAT2 antibody?

Verifying STAT2 antibody specificity is crucial for reliable experimental results. The most stringent method involves using STAT2 knockout cell lines as negative controls. According to the search results, multiple studies have demonstrated this approach:

• Knockout cell line validation: Western blot analysis using STAT2 knockout HeLa cell lines showed absence of the characteristic 110 kDa band that was clearly visible in parental cell lines .

• Complementary approach: Restoration of STAT2 expression in STAT2-deficient cells should restore antibody recognition .

• Isoform specificity: Validate whether the antibody recognizes both long and short STAT2 isoforms by analyzing cells known to express specific isoforms .

• Cross-reactivity testing: Test the antibody against cell lysates from multiple cell lines and against other STAT family proteins, particularly STAT1 which often interacts with STAT2 .

What are the typical applications for STAT2 antibodies in research?

STAT2 antibodies are employed in multiple research applications, with varying optimal dilutions:

The appropriate application depends on your specific research question. For example, Western blot is ideal for detecting STAT2 phosphorylation status, while IHC provides spatial information about STAT2 expression in tissue contexts .

How do different phosphorylation states of STAT2 affect antibody recognition?

STAT2 undergoes multiple phosphorylation events that can affect antibody recognition and signaling outcomes:

• Tyrosine phosphorylation: The classic activation mechanism following IFN stimulation. Antibodies specific to phosphorylated tyrosine residues (pY-STAT2) are crucial for monitoring STAT2 activation status in signaling studies .

• Serine/threonine phosphorylation: Multiple regulatory sites have been identified:

  • S734 phosphorylation negatively regulates antiviral responses by limiting expression of certain ISGs

  • T404 phosphorylation by IKK-ε influences STAT1-STAT2 dimer configuration and function

When selecting antibodies, researchers should consider:

• Whether they need to detect total STAT2 or specific phosphorylated forms

• The time course of different phosphorylation events (they follow different kinetics)

• The interdependence of phosphorylation events (e.g., S734 phosphorylation depends on prior tyrosine phosphorylation)

For experiments focusing on STAT2 activation mechanisms, using both total STAT2 and phospho-specific antibodies in parallel provides the most comprehensive analysis of signaling events.

What is the functional relationship between STAT1 and STAT2, and how does this impact experimental design?

The STAT1-STAT2 relationship is complex and has significant implications for experimental design:

• Constitutive interaction: STAT2 constitutively binds to STAT1 even in unstimulated cells, but not to STAT3, via a conserved interface .

• Dual roles: STAT2 can both activate and inhibit STAT1 function:

  • As part of ISGF3 complex in type I IFN signaling (activating)

  • By preventing nuclear translocation of STAT1 in response to IFN-γ, IL-6, and IL-27 (inhibitory)

• Dependency relationship: STAT1 is required for the C-STAT2 interaction, and expression of STAT1 enhances the inhibitory effect of the C protein on STAT2 activation .

Experimental design considerations:

• When studying STAT1-dependent pathways, consider the inhibitory effects of STAT2

• Use both STAT1 and STAT2 antibodies in co-immunoprecipitation experiments to detect interactions

• In knockout studies, remember that STAT2 deficiency can affect STAT1 expression levels

• Consider using the STAT2-L82A mutant, which cannot bind STAT1, to differentiate STAT1-dependent and independent functions of STAT2

How can STAT2 antibodies be used to investigate viral evasion of immune responses?

Viruses have evolved mechanisms to evade type I IFN responses, often targeting STAT2. Antibodies are essential tools for studying these evasion strategies:

• Detecting STAT2 degradation: Some viruses (like PIV5) target STAT2 for proteasomal degradation. Western blotting with STAT2 antibodies can quantify this degradation .

• Monitoring phosphorylation inhibition: Sendai virus C protein blocks STAT2 tyrosine phosphorylation. Phospho-specific STAT2 antibodies can reveal this inhibition mechanism .

• Subcellular localization studies: Using STAT2 antibodies in immunofluorescence can show altered localization patterns during viral infection.

• Virus-induced modifications: Special antibodies like those against T404-phosphorylated STAT2 can detect virus-induced phosphorylation by IKK-ε, revealing how viruses manipulate STAT2 function .

Research approach:

• Compare STAT2 levels, phosphorylation, and localization between mock-infected and virus-infected cells

• Use time-course experiments to track the kinetics of STAT2 targeting

• Combine with viral mutants to identify specific viral proteins responsible for STAT2 antagonism

What role does STAT2 play in dendritic cell function and how can antibodies help investigate this?

STAT2 has recently been identified as crucial for dendritic cell (DC) function beyond traditional type I IFN signaling:

• Constitutive expression: STAT2 is highly expressed in murine DCs constitutively, suggesting cell-intrinsic STAT2-dependent responses in DCs .

• Antitumor immunity: STAT2 in conventional DCs (cDCs) is critical for host IFN-I signals by shaping cytotoxic T lymphocyte (CTL) responses against tumors. Clinical correlation exists between cDC markers and STAT2 expression associated with better survival in human metastatic melanoma .

• TLR-induced activation: cDCs require STAT2 to respond to TLR ligands and upregulate costimulatory molecules, interferon-stimulated genes (ISGs), and pro-inflammatory cytokine IL-12 .

Experimental approaches with STAT2 antibodies:

• Flow cytometry to assess DC activation markers in relation to STAT2 expression

• Western blotting to monitor STAT2 phosphorylation following TLR stimulation

• IHC of tumor samples to correlate STAT2 expression in infiltrating DCs with patient outcomes

• ChIP assays to identify STAT2 binding sites in DC genomes

What are the optimal conditions for Western blot analysis using STAT2 antibodies?

Based on published protocols and manufacturer recommendations:

The search results show specific examples of successful Western blot protocols:

• R&D Systems protocol used 0.5 μg/mL of Goat Anti-Human STAT2 antibody with HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017)

• Proteintech recommends dilutions of 1:500-1:1000 for their STAT2 antibody (51075-2-AP)

For phosphorylated STAT2 detection, additional considerations apply:

• Quick sample processing to preserve phosphorylation status

• Inclusion of phosphatase inhibitors in lysis buffers

• Use of specific anti-pY-STAT2 antibodies (e.g., Upstate Biotechnology #07-224)

How should I design experiments to study STAT2's dual roles in type I and type II interferon signaling?

STAT2 has complex roles in both promoting type I IFN signaling and inhibiting type II IFN (IFN-γ) signaling. A comprehensive experimental design should:

• Compare wild-type and STAT2-deficient systems:

  • Use STAT2 knockout cell lines or STAT2-specific siRNA

  • Alternatively, use the STAT2-L82A mutant that cannot bind STAT1

• Measure both immediate signaling events and downstream effects:

  • Early events: STAT1/STAT2 phosphorylation, nuclear translocation, DNA binding

  • Downstream effects: ISG expression, antiviral protection, immune activation

• Study multiple time points:

  • STAT2 inhibition of STAT1 nuclear import occurs rapidly

  • Some effects on transcription may require longer incubation

• Include relevant controls and combinations:

  • Type I IFN alone (IFN-α or IFN-β)

  • Type II IFN alone (IFN-γ)

  • Sequential or simultaneous treatment with both IFN types

  • Additional cytokines (IL-6, IL-27) that activate STAT1

• Readouts to quantify:

  • Western blot: total and phosphorylated STAT1/STAT2

  • Confocal microscopy: nuclear translocation

  • EMSA: DNA-binding activity

  • qRT-PCR or RNA-seq: transcriptional responses

  • Functional assays: antiviral protection, immune activation

What controls should be included when validating a new STAT2 antibody?

A comprehensive validation protocol for STAT2 antibodies should include:

• Positive controls:

  • Cell lines known to express STAT2 (e.g., K562, Daudi, HeLa)

  • IFN-α stimulated cells (increases phospho-STAT2)

  • Recombinant STAT2 protein (if available)

• Negative controls:

  • STAT2 knockout cell line (gold standard)

  • Cell lysates depleted of STAT2 via immunoprecipitation

  • Tissues from STAT2 knockout animals

• Specificity controls:

  • Other STAT family members (especially STAT1)

  • Cell lines expressing different STAT2 isoforms

  • Blocking with immunizing peptide (for polyclonal antibodies)

• Application-specific controls:

  • For WB: Molecular weight markers

  • For IHC: Isotype control antibodies

  • For IF: Secondary antibody-only control

• Cross-species reactivity testing:

  • If claiming multi-species reactivity, test each species

  • Be aware that human STAT2 shares only 73% identity with rat and 65% with mouse

How can I detect and quantify STAT2 phosphorylation at different regulatory sites?

STAT2 function is regulated by phosphorylation at multiple sites, each requiring specific detection methods:

• Tyrosine phosphorylation (activation):

  • Anti-pY-STAT2 antibodies (e.g., Upstate Biotechnology #07-224)

  • Expected timing: Rapid (minutes) after IFN-α stimulation

  • Key site: Y690 (primary activation site)

• Serine phosphorylation (regulatory):

  • Site-specific antibodies for S734

  • Different kinetics from tyrosine phosphorylation

  • Functional effect: Negatively regulates antiviral responses

• Threonine phosphorylation (virus-induced):

  • Specific antibodies against T404-phosphorylated STAT2

  • Induced by IKK-ε during viral infection

  • Affects STAT1-STAT2 dimer configuration

Experimental approach:

• Use phospho-specific antibodies in Western blot

• Include phosphatase inhibitors in lysis buffers

• Perform time-course experiments (different sites have different kinetics)

• Use phosphatase treatment of duplicate samples as controls

• Consider mass spectrometry for comprehensive phosphorylation profiling

• Use kinase inhibitors to confirm the responsible kinases

Why might I observe inconsistent STAT2 detection in Western blots?

Inconsistent STAT2 detection can result from several factors:

• Sample preparation issues:

  • Phosphorylation status deteriorates rapidly; process samples quickly

  • Include protease and phosphatase inhibitors

  • Ensure complete protein denaturation (STAT2 is a large protein)

• Technical considerations:

  • STAT2 is a large protein (~113 kDa) that requires efficient transfer conditions

  • Longer transfer times or specialized transfer methods may be necessary

  • Reducing conditions are critical (all cited protocols specify reducing conditions)

• Biological factors:

  • Varying STAT2 expression levels between cell types

  • Possible interference from STAT1 binding

  • Degradation during viral infection if studying viral evasion mechanisms

• Antibody-specific factors:

  • Some antibodies recognize specific phosphorylation states

  • Epitope may be masked by protein-protein interactions

  • Batch-to-batch variability in polyclonal antibodies

Troubleshooting steps:

• Include positive controls (e.g., IFN-α stimulated cells)

• Test multiple STAT2 antibodies targeting different epitopes

• Optimize protein extraction, transfer conditions, and blocking reagents

• For phospho-specific detection, stimulate cells with appropriate cytokines

How can I optimize STAT2 immunohistochemistry in tissue samples?

Immunohistochemistry (IHC) with STAT2 antibodies requires specific optimization:

• Antigen retrieval is critical:

  • TE buffer pH 9.0 is recommended for STAT2 IHC

  • Alternative: citrate buffer pH 6.0 has been used successfully

• Antibody concentration:

  • Higher concentrations than WB are typically needed (1:20-1:200 dilution range)

  • Titrate to determine optimal concentration for your specific tissue

• Controls:

  • Positive control tissues: human breast cancer and lung cancer tissues have been validated

  • Negative controls: STAT2-deficient tissues or isotype control antibodies

• Detection systems:

  • HRP-based systems are commonly used

  • Consider tyramide signal amplification for low abundance detection

• Blocking and reducing background:

  • Use protein block containing both albumin and serum

  • Consider additional avidin/biotin blocking if using biotin-based detection

Optimization strategy:

• Test multiple antibody concentrations

• Compare different antigen retrieval methods

• Adjust incubation times (overnight at 4°C often yields cleaner results)

• For dual IHC (e.g., with phospho-specific antibodies), carefully select compatible detection systems

How can I distinguish between research artifacts and true biological effects when studying STAT2?

Distinguishing between artifacts and true biological effects requires systematic controls:

• For phosphorylation studies:

  • Include both positive controls (IFN-α stimulated cells) and negative controls (unstimulated cells)

  • Use multiple time points (phosphorylation events have specific kinetics)

  • Compare results from multiple phospho-specific antibodies

  • Confirm with phosphatase treatment of duplicate samples

• For knockout/knockdown experiments:

  • Use multiple siRNAs or shRNAs targeting different regions of STAT2

  • Include rescue experiments with exogenous STAT2 expression

  • Be aware that STAT2 deficiency can affect STAT1 levels in some cell types

• For protein-protein interaction studies:

  • Perform reciprocal co-immunoprecipitations

  • Include stimulus-dependent controls

  • Consider proximity ligation assays for in situ detection

  • Compare multiple antibodies targeting different epitopes

• For functional studies:

  • Use multiple readouts (e.g., gene expression, protein levels, functional assays)

  • Compare results across different cell types

  • Consider genetic approaches (CRISPR/Cas9) alongside antibody-based methods

  • Use the STAT2-L82A mutant that selectively disrupts STAT1 binding

How are STAT2 antibodies being used to study the role of dendritic cells in anti-tumor immunity?

Recent research has revealed critical roles for STAT2 in dendritic cell-mediated anti-tumor immunity:

• Clinical correlations: STAT2 expression in conventional dendritic cells (cDCs) correlates with better survival in human metastatic melanoma .

• Mechanistic insights: STAT2 in CD11c+ cDCs is essential for:

  • Activation of both CD8a+ cDCs and CD11b+ cDCs

  • Antigen cross-presentation for robust T cell killing responses

  • Response to therapeutic IFNβ in tumor models

• Methodological approaches using STAT2 antibodies:

  • Flow cytometry to assess DC activation markers following stimulation

  • Confocal microscopy to track STAT2 nuclear translocation

  • ChIP-seq to identify STAT2 binding sites in DC genomes

  • Western blotting to monitor STAT2 phosphorylation status

• Targeted cell-specific studies: Using Cre-lox systems for DC-specific STAT2 deletion combined with antibody detection methods has revealed that STAT2 in cDCs is critical for host IFN-I signals by sculpting CTL responses against tumors .

This emerging field highlights the need for rigorous experimental design combining genetic approaches with sensitive and specific STAT2 antibody-based detection methods.

What are the latest findings on STAT2's role in regulating other STAT proteins?

Recent research has uncovered STAT2 as a pervasive cytokine regulator through its inhibition of STAT1 in multiple signaling pathways:

• Constitutive binding: STAT2 constitutively binds to STAT1 (but not STAT3) via a conserved interface even in unstimulated cells .

• Selective inhibition mechanism: Unphosphorylated STAT2 dimerizes with activated STAT1, creating semiphosphorylated dimers incapable of importin-α binding, preventing nuclear translocation specifically of STAT1 in response to IFN-γ, IL-6, and IL-27 .

• Functional consequences:

  • Attenuated IFN-γ responses (MHC expression, senescence, antiparasitic immunity)

  • Shifted transcriptional output of IL-27 from STAT1 to STAT3

  • Altered balance between pro- and anti-inflammatory responses

• Experimental tools: The STAT2-L82A mutant, which cannot bind STAT1, has enabled researchers to dissociate STAT2's activating and inhibitory effects on STAT1 .

This new understanding has significant implications for interpreting experiments involving STAT2 antibodies, particularly when studying cytokine signaling networks and immune regulation.

How can phospho-specific STAT2 antibodies advance our understanding of signaling regulation?

Phospho-specific STAT2 antibodies are revealing complex regulatory mechanisms:

• S734 phosphorylation: Recently identified as negatively regulating the antiviral effects of type I interferons by limiting the expression of a select subset of antiviral ISGs .

• T404 phosphorylation: Induced by virus infection through IKK-ε kinase activity, affecting STAT1-STAT2 dimer configuration and function .

• Integration of multiple signals: Phospho-specific antibodies reveal how different modification patterns integrate multiple upstream signals.

• Kinetics and dependencies: S734 phosphorylation displays different kinetics than tyrosine phosphorylation and is dependent on prior tyrosine phosphorylation and JAK1 activity .

• Virus-host interactions: Specific antibodies against phosphorylated T404 have revealed how viruses manipulate STAT2 function through IKK-ε and TBK1 .

These tools are advancing our understanding of the molecular mechanisms underlying STAT2's diverse functions and how these mechanisms are exploited during viral infection or dysregulated in disease.

What contradictions in STAT2 research might be explained by methodological differences in antibody-based detection?

Several apparent contradictions in STAT2 research might be explained by methodological differences:

• STAT1 dependence in STAT2 activation:

  • Some studies suggest STAT2 requires STAT1 for activation

  • Others show STAT1-independent STAT2 functions

  • Explanation: Different antibodies may detect different conformational states of STAT2

• STAT2 deficiency phenotypes:

  • Global STAT2 knockout shows profound antiviral defects

  • Cell-specific knockouts show more nuanced phenotypes

  • Explanation: Different antibodies and detection methods may have varying sensitivities

• Type I IFN-dependent vs. independent functions:

  • Some studies show strict IFN-dependence

  • Others suggest IFN-independent STAT2 activities

  • Explanation: Antibodies targeting different STAT2 epitopes may detect different protein complexes

• Discrepancies in phosphorylation studies:

  • Variable effects reported for specific phosphorylation sites

  • Explanation: Timing of sample collection is critical; phosphorylation events have distinct kinetics

Researchers should carefully consider:

• Specificity of antibodies used (epitope location, cross-reactivity)

• Timing of analyses (phosphorylation kinetics vary)

• Technical differences in sample preparation

• Cell type-specific differences in STAT2 function and expression

What are the key considerations for selecting and validating STAT2 antibodies for specific research applications?

When selecting and validating STAT2 antibodies, researchers should consider:

• Research question specificity:

  • Total STAT2 vs. phosphorylated forms

  • Subcellular localization studies vs. expression level analysis

  • Protein-protein interaction studies vs. functional analyses

• Technical validation requirements:

  • Knockout/knockdown controls are essential

  • Cross-reactivity with other STAT family members must be assessed

  • Species reactivity should be experimentally verified

  • Application-specific validation (WB, IF, IHC, etc.)

• Biological context considerations:

  • Cell type-specific expression levels

  • Stimulus-dependent modifications

  • Potential interference from STAT1 binding

  • Isoform specificity

• Strategic antibody selection:

  • Use multiple antibodies targeting different epitopes for critical findings

  • Consider the specific epitope location and its functional relevance

  • Balance between monoclonal specificity and polyclonal coverage

  • Verify commercial validation data with independent experiments