Phospho-STAT1 (S727) Recombinant Monoclonal Antibody

What is STAT1 and what role does S727 phosphorylation play?

STAT1 (Signal Transducer and Activator of Transcription 1) functions as a critical transcription factor mediating cellular responses to interferons (IFNs), cytokines, and growth factors. STAT1 requires phosphorylation at two key sites for optimal function: tyrosine 701 (Y701) and serine 727 (S727). While Y701 phosphorylation is essential for STAT1 dimerization and nuclear translocation, S727 phosphorylation is required for full transcriptional activity and biological function .

The phosphorylation at S727 represents a final activation step that occurs after STAT1 has been assembled into chromatin-associated transcriptional complexes. This mechanism ensures that only properly localized and assembled STAT1 molecules achieve full activation, providing an additional layer of regulation in the signaling pathway . S727 phosphorylation has also been proposed to potentially accelerate the disassembly of transcriptional complexes after mRNA synthesis initiation, enabling more rapid cycling of STAT1 activation .

How is Phospho-STAT1 (S727) Recombinant Monoclonal Antibody produced?

The production of Phospho-STAT1 (S727) recombinant monoclonal antibody involves several sophisticated biotechnological steps:

• Animal immunization with a synthesized peptide derived from human phospho-STAT1 (S727)

• Isolation of B cells from immunized mice

• Selection of positive B cells followed by single clone identification

• PCR amplification of light and heavy chains of the antibody

• Insertion of amplified chains into a plasmid vector to create a recombinant vector

• Transfection of the vector into host cells to express the antibody

• Purification of the antibody from cell culture supernatant using affinity chromatography

This recombinant approach ensures high specificity and reproducibility compared to traditional monoclonal antibody production methods, making it particularly valuable for detecting specific phosphorylation states of STAT1 .

What applications is the Phospho-STAT1 (S727) antibody validated for?

The Phospho-STAT1 (S727) antibody has been validated for multiple research applications:

These applications enable researchers to investigate the phosphorylation status of STAT1 at S727 in various experimental contexts, from protein expression levels to cellular localization .

How does STAT1 S727 phosphorylation differ between interferon and stress signaling pathways?

The regulation of STAT1 S727 phosphorylation follows distinct mechanisms depending on the stimulus:

In interferon signaling:

• IFN-induced S727 phosphorylation requires:

  • Y701 phosphorylation

  • Nuclear translocation

  • Assembly into chromatin-associated transcriptional complexes

• An intact SH2 domain is essential for this process

• The R602K mutation (binding-deficient SH2 domain) prevents S727 phosphorylation in response to IFN-γ

• Y701F mutation also blocks IFN-induced S727 phosphorylation

In stress-induced signaling (e.g., UV irradiation):

• S727 phosphorylation occurs independently of:

  • Y701 phosphorylation

  • SH2 domain integrity

  • Nuclear localization

• p38 MAPK pathway is required for UV-induced S727 phosphorylation

• Both STAT1-R602K and STAT1-Y701F mutants show normal S727 phosphorylation in response to UV irradiation or anisomycin (p38 MAPK activator)

This dual regulation mechanism enables STAT1 to respond differently to cytokines versus cellular stress, with distinct downstream consequences for gene expression .

What is the relationship between chromatin association and STAT1 S727 phosphorylation?

Research has revealed a critical link between chromatin association and S727 phosphorylation of STAT1:

• Chromatin recruitment is required for IFN-γ-induced S727 phosphorylation

• STAT1 mutants with diminished DNA binding ability (K336A, K544A/E545A, and N460A) show reduced S727 phosphorylation in response to IFN-γ

• IFN-β can restore S727 phosphorylation of these mutants through formation of the ISGF3 complex (STAT1/STAT2/IRF9)

• In the ISGF3 complex, IRF9 serves as the main DNA binding subunit, allowing chromatin association despite mutations in STAT1's DNA binding domain

The requirement for chromatin association represents a quality control mechanism that ensures only properly assembled STAT1 transcriptional complexes achieve full activation. This mechanism restricts the final activation step (S727 phosphorylation) to chromatin-tethered transcription factors, preventing inappropriate activation of soluble nuclear STAT1 .

How does S727 phosphorylation affect STAT1 target gene expression?

The impact of S727 phosphorylation on STAT1-mediated gene expression is complex and gene-specific:

• Mutation of S727 differentially affects IFN-γ target genes at both basal and induced expression levels

• Particularly strong effects have been observed for genes such as:

  • GBP1 (Guanylate Binding Protein 1)

  • TAP1 (Transporter Associated with Antigen Processing 1)

• Different promoters exhibit varying requirements for S727 phosphorylation

• The effects may depend on:

  • Promoter architecture

  • Co-factor requirements

  • Chromatin accessibility at specific loci

These findings suggest that S727 phosphorylation contributes to signaling specificity by differentially regulating subsets of STAT1 target genes. This selective regulation allows for fine-tuning of the interferon response based on context and cell type .

What are the optimal conditions for detecting STAT1 S727 phosphorylation in Western blot experiments?

To achieve optimal detection of STAT1 S727 phosphorylation by Western blot:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors to preserve phosphorylation status

  • Include both positive controls (IFN-γ or IFN-β treated cells) and negative controls (untreated cells)

  • Consider including UV-irradiated samples as an alternative positive control that operates through a different pathway

• SDS-PAGE and transfer:

  • Use 7-8% gels for optimal resolution of the ~91 kDa STAT1 protein

  • Ensure complete transfer to PVDF or nitrocellulose membranes

• Antibody incubation:

  • Recommended dilution range: 1:500-1:5000 for Western blot applications

  • Block membranes thoroughly to reduce background

  • Consider overnight primary antibody incubation at 4°C for maximum sensitivity

• Detection considerations:

  • STAT1 has two isoforms (α and β); the phospho-S727 antibody detects only the α isoform (91 kDa) as S727 is located in the C-terminal transactivation domain absent in the β isoform (84 kDa)

  • Stripping and reprobing with total STAT1 antibody allows calculation of the phospho/total ratio

These methodological details are essential for obtaining reliable and reproducible results when studying STAT1 S727 phosphorylation dynamics .

How can researchers validate the specificity of phospho-STAT1 (S727) antibody in their experimental systems?

Validating antibody specificity is crucial for accurate interpretation of results. For phospho-STAT1 (S727) antibody, consider these validation approaches:

• Phosphatase treatment control:

  • Treat half of your positive control sample with lambda phosphatase before immunoblotting

  • Loss of signal confirms phospho-specificity

• Peptide competition assay:

  • Pre-incubate the antibody with the phosphopeptide immunogen

  • Signal reduction/elimination confirms specificity for the phospho-epitope

• Genetic validation:

  • Use STAT1-deficient cells (e.g., STAT1-/- fibroblasts) as negative controls

  • Compare STAT1 wild-type with S727A mutant-expressing cells

  • The S727A mutant should show no reactivity with the phospho-antibody

• Stimulus-dependent phosphorylation:

  • Treat cells with known inducers of S727 phosphorylation (IFN-γ, IFN-β)

  • Include both short (15-30 min) and long (1-2 h) time points

  • Compare with stress inducers like UV or anisomycin that activate different pathways

These validation steps ensure that the observed signals genuinely represent S727-phosphorylated STAT1, minimizing the risk of misinterpreting experimental data .

What are the key considerations for immunohistochemical detection of phospho-STAT1 (S727)?

For successful immunohistochemical detection of phospho-STAT1 (S727) in tissue sections:

• Fixation and antigen retrieval:

  • Phospho-epitopes can be sensitive to overfixation

  • Use freshly prepared 4% paraformaldehyde or 10% neutral buffered formalin

  • Optimize antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Heating-based retrieval must balance epitope exposure against potential dephosphorylation

• Protocol optimization:

  • Recommended dilution: 1:50-1:200 for IHC applications

  • Include phosphatase inhibitors in all buffers

  • Consider signal amplification systems for low abundance targets

• Controls:

  • Include known positive tissues (e.g., IFN-stimulated lymphoid tissues)

  • Use adjacent sections with phospho-independent STAT1 antibody to compare distribution

  • Consider peptide competition controls

• Interpretation:

  • Active phospho-STAT1 (S727) should show primarily nuclear localization

  • Evaluate both staining intensity and subcellular localization

  • Counterstain nuclei to facilitate assessment of nuclear translocation

These considerations help maintain phospho-epitope integrity throughout the IHC procedure and ensure accurate visualization of phospho-STAT1 (S727) in tissues .

How can researchers address inconsistent results in STAT1 S727 phosphorylation detection?

Inconsistent results when detecting STAT1 S727 phosphorylation may stem from several sources. Here are troubleshooting strategies:

• For weak or absent signals:

  • Verify stimulus effectiveness (use positive control like p-Y701 detection)

  • Ensure appropriate timepoint (S727 phosphorylation typically peaks 30-60 min after stimulation)

  • Check phosphatase inhibitor effectiveness in all buffers

  • Optimize antibody concentration and incubation conditions

• For high background or non-specific signals:

  • Increase blocking stringency

  • Optimize antibody dilution

  • Reduce primary antibody incubation time or temperature

  • Consider alternative detection systems with lower background

• For contradictory results between techniques:

  • Different techniques have varying sensitivity thresholds

  • IF may detect localized nuclear phospho-STAT1 more effectively than WB of whole cell lysates

  • Consider cell type-specific differences in phosphorylation kinetics

• For temporal inconsistencies:

  • S727 phosphorylation follows Y701 phosphorylation and nuclear translocation

  • Examine the complete time course (15 min to 4 hours)

  • Cell type differences affect phosphorylation kinetics

By systematically addressing these potential issues, researchers can improve the consistency and reliability of phospho-STAT1 (S727) detection across experiments .

How do different interferon types affect STAT1 S727 phosphorylation patterns?

Different interferon types induce distinctive patterns of STAT1 S727 phosphorylation:

• Type I interferons (IFN-α, IFN-β):

  • Activate both STAT1:STAT1 homodimers and STAT1:STAT2:IRF9 (ISGF3) complexes

  • Can rescue S727 phosphorylation of STAT1 DNA-binding mutants through ISGF3 formation

  • IRF9 in ISGF3 provides chromatin association capability

  • Induce a broader range of genes than type II interferons

• Type II interferon (IFN-γ):

  • Primarily activates STAT1:STAT1 homodimers

  • S727 phosphorylation strictly depends on STAT1's own DNA binding capability

  • STAT1 DNA-binding mutants show poor S727 phosphorylation response to IFN-γ

  • Cannot compensate for defects in STAT1 chromatin association

• Comparative dynamics:

  • IFN-γ often induces more potent but transient S727 phosphorylation

  • IFN-β may induce more sustained S727 phosphorylation

  • The kinetics reflect different downstream kinase activation patterns

  • Cell type-specific factors significantly influence these patterns

These differences highlight the complexity of interferon signaling and explain why experimental outcomes may vary depending on the specific interferon type used .

What impact do mutations in STAT1 have on detecting S727 phosphorylation?

Mutations in STAT1 can significantly affect S727 phosphorylation and its detection:

• Tyrosine phosphorylation site mutation (Y701F):

  • Prevents IFN-induced S727 phosphorylation

  • Still permits stress-induced (UV, anisomycin) S727 phosphorylation

  • Demonstrates the requirement for Y701 phosphorylation in the IFN pathway

• SH2 domain mutation (R602K):

  • Blocks Y701 phosphorylation and subsequent nuclear translocation

  • Prevents IFN-induced S727 phosphorylation

  • Stress-induced S727 phosphorylation remains intact

• DNA binding mutations (K336A, K544A/E545A, N460A):

  • Allow nuclear translocation but reduce chromatin association

  • Show poor S727 phosphorylation in response to IFN-γ

  • S727 phosphorylation can be rescued by IFN-β through ISGF3 complex formation

• Nuclear localization mutations (N-terminal deletion Δ27, L407A):

  • Prevent nuclear accumulation despite Y701 phosphorylation

  • Block IFN-induced S727 phosphorylation

  • Adding an SV40 NLS can restore nuclear localization but not S727 phosphorylation without Y701 phosphorylation

These mutation studies have been instrumental in deciphering the mechanisms regulating STAT1 S727 phosphorylation and should be considered when interpreting experimental results .

What new insights have emerged about the kinases responsible for STAT1 S727 phosphorylation?

Recent research has revealed complex regulation of kinases responsible for STAT1 S727 phosphorylation:

• Interferon-induced pathway:

  • The kinase responsible for IFN-induced S727 phosphorylation remains incompletely identified

  • Evidence suggests this kinase associates with chromatin-bound STAT1 complexes

  • The mechanism ensures phosphorylation occurs only after proper assembly into transcriptional complexes

• Stress-induced pathway:

  • p38 MAPK is clearly established as the primary kinase for stress-induced S727 phosphorylation

  • This pathway operates independently of Y701 phosphorylation and nuclear localization

  • p38 MAPK can be activated by UV irradiation, anisomycin, and other cellular stresses

• Context-specific regulation:

  • S727 phosphorylation of STAT1 in IFN-γ-treated mouse fibroblasts occurs without requirement for p38 MAPK, ERK1/2, or JNK

  • In contrast, STAT3's PMS727P motif is phosphorylated by different stimuli and signaling pathways

  • The C-terminus of STATs contributes to signaling specificity by linking individual STATs to different serine kinase pathways

These findings suggest a specialized kinase machinery for IFN-induced STAT1 S727 phosphorylation that is distinct from stress-activated pathways, highlighting the precise regulation of this modification .

How does the dual phosphorylation mechanism of STAT1 contribute to signaling specificity?

The dual phosphorylation mechanism of STAT1 (Y701 and S727) provides several layers of signaling specificity:

• Sequential activation control:

  • Y701 phosphorylation serves as a prerequisite for nuclear translocation

  • S727 phosphorylation represents a final activation step after chromatin association

  • This sequential process ensures only properly localized STAT1 achieves full activation

• Differential gene regulation:

  • S727 phosphorylation differentially affects IFN-γ target genes

  • Some genes (e.g., GBP1, TAP1) are strongly dependent on S727 phosphorylation

  • Other genes show less dependency, allowing graduated responses

• Pathway-specific activation:

  • Chromatin association requirement restricts IFN-induced S727 phosphorylation to transcriptionally engaged STAT1

  • Stress-induced S727 phosphorylation follows different rules, allowing independent responses to cellular stress

  • The C-terminal domain contributes to pathway specificity (demonstrated by STAT3 C-terminus transfer experiments)

• Potential role in signal termination:

  • S727 phosphorylation may accelerate disassembly of transcriptional complexes

  • This could facilitate more rapid cycling of STAT1 to the receptor

  • A role in nuclear export has been proposed, suggesting dual functions in activation and termination

This sophisticated regulation allows cells to fine-tune their responses to different stimuli, generating context-appropriate gene expression programs .

What emerging applications of Phospho-STAT1 (S727) antibodies show promise for future research?

Several emerging applications of Phospho-STAT1 (S727) antibodies hold promise for advancing our understanding of STAT1 biology:

• Single-cell analysis techniques:

  • Combining phospho-STAT1 (S727) antibodies with single-cell technologies

  • Investigating cell-to-cell variability in STAT1 activation

  • Correlating S727 phosphorylation with transcriptional output at single-cell resolution

• Chromatin immunoprecipitation applications:

  • Using phospho-specific ChIP to map genomic localization of S727-phosphorylated STAT1

  • Comparing occupancy patterns between total STAT1 and phospho-S727 STAT1

  • Correlating S727 phosphorylation status with co-factor recruitment at specific promoters

• Therapeutic relevance in disease models:

  • Investigating S727 phosphorylation in interferon-resistant states

  • Exploring the role of STAT1 S727 phosphorylation in autoimmune conditions

  • Assessing phospho-STAT1 (S727) as a biomarker in cancer immunotherapy response

• Structure-function relationships:

  • Using phospho-specific antibodies to understand conformational changes induced by S727 phosphorylation

  • Investigating how S727 phosphorylation affects STAT1 interactions with chromatin and transcriptional machinery

  • Developing biosensors based on phospho-specific antibodies to monitor STAT1 activation dynamics in living cells

These applications will help elucidate the complex roles of STAT1 S727 phosphorylation in normal physiology and disease states, potentially leading to new therapeutic approaches .