SMAD1 (Ab-206) Antibody

What is SMAD1 protein and what role does the phosphorylated S206 site play?

SMAD1 functions as a signal transducer and transcriptional modulator in multiple signaling pathways. It is also known as Mothers Against Decapentaplegic homolog 1, hSMAD-3, JV4-1, Transforming growth factor-Beta-Signaling Protein 1, or BSP1 . As a transcriptional modulator, SMAD1 is activated by BMP (Bone Morphogenetic Protein) type 1 receptor kinase, classifying it as a receptor-regulated SMAD (R-SMAD) .

The phosphorylation at serine 206 (pS206) occurs as part of agonist-induced phosphorylation in the interdomain linker region. This phosphorylation is primarily mediated by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes . The pS206 modification plays a crucial dual role in both Smad transcriptional activation and protein turnover. This site is specifically targeted by ERK in response to mitogenic growth factors, and can be experimentally induced by treating cells with EGF or in cancer cells where Ras is activated .

How does SMAD1 phosphorylation at S206 differ from other phosphorylation sites?

SMAD1 contains multiple phosphorylation sites that serve distinct functions. Besides S206 in the linker region, other prominent phosphorylation sites include S187, S195, S210, and S214 . These sites are regulated by different kinases and have specific roles:

• The C-terminal SXS motif is phosphorylated by BMP receptor kinases, initiating SMAD1 activation .

• The linker region sites (including S206) are phosphorylated by CDK8/9 following the initial C-terminal phosphorylation .

• Additional linker phosphorylation by GSK3 at sites like T202 and S210 occurs subsequently .

What distinguishes S206 phosphorylation is its specific role in recruiting cofactors like YAP to the phosphorylated linker sites, which supports SMAD1-dependent transcription and is required for BMP suppression of neural differentiation in mouse embryonic stem cells . Unlike the SXS motif phosphorylation, which primarily initiates signaling, S206 phosphorylation serves both to enhance transcriptional activity and to mark the protein for eventual degradation .

What are the primary applications of SMAD1 (Ab-206) Antibody in research?

The SMAD1 (Ab-206) Antibody is primarily utilized in research applications including:

• Western Blotting (WB): For detecting phosphorylated SMAD1 at S206 in cell or tissue lysates, allowing researchers to monitor activation of this specific phosphorylation site .

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative assessment of SMAD1 phosphorylation levels in various experimental conditions .

• Chromatin Immunoprecipitation (ChIP): As demonstrated in research, antibodies against phospho-Ser206 of SMAD1 can be used to pull down DNA containing BMP responsive regions of genes like Id1 and Smad7, confirming the presence of phosphorylated SMAD1 at target gene promoters .

• Monitoring BMP and TGFβ signaling pathway activation: The antibody allows researchers to specifically track the phosphorylation status at S206, which serves as a marker for active BMP signaling and subsequent transcriptional events .

• Studying cross-talk between signaling pathways: Since S206 can be phosphorylated by both CDK8/9 (in response to BMP) and ERK (in response to mitogenic factors), this antibody enables the investigation of pathway interactions .

How can SMAD1 (Ab-206) Antibody be used to investigate the relationship between BMP signaling and cellular transcription mechanisms?

SMAD1 (Ab-206) Antibody provides a powerful tool for investigating the molecular link between BMP signaling and transcriptional regulation through several methodological approaches:

• Chromatin Occupancy Analysis: By combining ChIP with the pS206-specific antibody and sequencing (ChIP-seq), researchers can map genome-wide binding sites of phosphorylated SMAD1. Studies have shown that both anti-Smad1/5 antibody and antibody against phospho-Ser206 of Smad1 successfully pulled down DNA containing BMP responsive regions of Id1 and Smad7 genes in BMP-treated cells .

• Transcriptional Complex Assembly: The pS206 modification facilitates interactions with transcriptional cofactors. Immunoprecipitation with the Ab-206 antibody followed by mass spectrometry can identify novel interaction partners that are recruited specifically to the phosphorylated linker region.

• Real-time Transcriptional Dynamics: By combining the antibody with fluorescence microscopy techniques, researchers can visualize the temporal relationship between SMAD1 linker phosphorylation and target gene activation. The research shows that BMP signaling induces measurable changes in expression of target genes like Id1, which can be correlated with SMAD1 phosphorylation status .

• Kinase-Phosphatase Balance Assessment: Since CDK8/9 mediates the phosphorylation at S206, while phosphatases like SCP4 specifically target other sites, the Ab-206 antibody can help dissect the temporal dynamics of phosphorylation and dephosphorylation events that regulate transcriptional duration .

What are the structural implications of S206 phosphorylation on SMAD1 interactions with WW domain proteins?

The phosphorylation of S206 in the SMAD1 linker region creates a specific binding interface that is recognized by WW domain-containing proteins, with significant structural consequences:

• YAP WW Domain Interaction: The phosphorylated S206 and P207 side chains are accommodated in the aromatic cavity formed by Tyr188 and Trp199 in the YAP WW1 domain . Isothermal titration calorimetry (ITC) experiments have shown that YAP WW1-WW2 segments bind unphosphorylated SMAD1 peptides with low affinity, but phosphorylation at S206 significantly enhances this interaction .

• Binding Mode Specificity: The YAP-SMAD1 complex involves recognition of the SMAD1 PY motif by the YAP WW2 domain, while the CDK-phosphorylated site pS206 is specifically recognized by the WW1 domain . This dual recognition mechanism ensures specificity and stability of the interaction.

• Conformational Changes: Upon binding to WW domain proteins, the SMAD1 linker region adopts an extended conformation. While this has been more extensively characterized for other phosphorylation sites (like pS210-pS214 binding to Smurf1), the pS206 site likely induces similar conformational adaptations when interacting with its binding partners .

• Impact on Protein-Protein Interaction Networks: The phosphorylation of S206 reconfigures SMAD1's interaction landscape, facilitating recruitment of transcriptional co-activators while also eventually marking the protein for ubiquitination and degradation, thereby creating a self-limiting signaling mechanism .

How can researchers troubleshoot non-specific binding or weak signals when using SMAD1 (Ab-206) Antibody?

When encountering challenges with SMAD1 (Ab-206) Antibody specificity or signal strength, researchers should consider these methodological approaches:

• Validation with Phosphatase Treatment: To confirm signal specificity, treat one sample with lambda phosphatase before immunoblotting. A genuine phospho-specific antibody will show signal reduction in the phosphatase-treated sample. Studies have shown that phosphatases like PPM1A effectively reduce phospho-SMAD1 levels, providing a useful positive control .

• Optimization of Signal Activation: Maximize phosphorylation at S206 by using appropriate BMP stimulation conditions. Research indicates that BMP2 treatment induces robust phosphorylation of the SMAD1 linker region . Consider using constitutively active BMP receptors like ALK3(Q233D) which has been shown to significantly increase P-SMAD1 levels .

• Blocking Non-specific Binding Sites: Use a combination of BSA (3-5%) and non-fat dry milk (3-5%) in your blocking buffer, as the different blocking agents can complement each other in reducing background.

• Cross-Reactivity Assessment: Be aware that while the antibody is designed for pS206 specificity, it may have varying degrees of cross-reactivity with other phosphorylated residues. Experimental evidence suggests that some phospho-specific antibodies recognize distinct sites - for example, SCP4 specifically dephosphorylates the SXS motif but not Ser-206 in the linker region, allowing researchers to distinguish between these phosphorylation events .

• Positive Control Implementation: Include a positive control of cells treated with agents known to induce S206 phosphorylation. Research shows that besides BMP, treatment with EGF or using cells with activated Ras can increase phosphorylation at this site through ERK pathway activation .

What are the optimal sample preparation methods for detecting phosphorylated SMAD1 at S206?

To achieve reliable detection of phosphorylated SMAD1 at S206, researchers should implement the following sample preparation protocol:

• Rapid Sample Collection and Processing: Phosphorylation states are dynamic and can change quickly. Minimize the time between cell harvesting and protein extraction, ideally keeping samples on ice throughout processing.

• Phosphatase Inhibitor Inclusion: Always include a comprehensive phosphatase inhibitor cocktail in lysis buffers. Studies have shown that phosphatases like SCP4 can specifically dephosphorylate SMAD proteins, potentially leading to false negatives if not properly inhibited .

• BMP Stimulation Optimization: For positive controls or experimental induction, stimulate cells with BMP2 (typically 25-100 ng/ml) for 1 hour, which has been demonstrated to effectively induce SMAD1 linker phosphorylation . Alternatively, transfection with constitutively active ALK3(Q233D) can be used to increase P-SMAD1 levels .

• Nuclear Fraction Enrichment: Since phosphorylated SMAD1 predominantly accumulates in the nucleus after BMP stimulation, nuclear fractionation can increase detection sensitivity. Evidence shows that flavopiridol can prevent ALP of nuclear SMAD1 in BMP-treated cells, indicating the nuclear localization of this phosphorylation event .

• Sample Denaturation Protocol: Use SDS sample buffer with 5% β-mercaptoethanol and heat at 95°C for 5 minutes to fully denature the protein and expose the phosphorylated epitope.

• Verification with Phosphatase Treatment: Process duplicate samples with and without lambda phosphatase treatment to verify antibody specificity to the phosphorylated form.

How can SMAD1 (Ab-206) Antibody be utilized in combination with other antibodies to study pathway crosstalk?

Multiplexed antibody approaches can reveal intricate signaling relationships between the BMP pathway and other cellular processes:

• Co-immunostaining Protocol: Combine SMAD1 (Ab-206) with antibodies against ERK pathway components (phospho-ERK1/2) to investigate dual regulation of S206 by both BMP and mitogenic signals. Research indicates that ERK can phosphorylate S206 in response to mitogenic growth factors, establishing a point of pathway convergence .

• Sequential Immunoprecipitation Strategy: First immunoprecipitate with SMAD1 (Ab-206) antibody, then perform a second immunoprecipitation with antibodies against potential interaction partners like YAP to isolate specific complex populations. Studies have shown that phosphorylated linker sites promote YAP recruitment to SMAD1, which supports SMAD1-dependent transcription .

• Pathway Inhibitor Studies: Combine antibody detection with pathway-specific inhibitors to distinguish between different sources of S206 phosphorylation:

  • CDK8/9 inhibitors (flavopiridol): Blocks BMP-induced linker phosphorylation

  • MEK inhibitors (U0126): Target ERK pathway-mediated phosphorylation

• Multiparameter Flow Cytometry: For single-cell analysis of pathway activation, combine SMAD1 (Ab-206) with antibodies against:

  • C-terminal phosphorylated SMAD1 (SXS motif)

  • YAP/TAZ (Hippo pathway components)

  • Cell cycle markers (since CDK8/9 activity may vary across the cell cycle)

• ChIP-reChIP Technique: First perform ChIP with SMAD1 (Ab-206), then re-ChIP the isolated material with antibodies against transcriptional cofactors to identify specific regulatory complexes at target genes. Research has shown that both anti-Smad1/5 antibody and an antibody against phospho-Ser206 of Smad1 successfully pulled down BMP-responsive genomic regions .

What controls should be included when using SMAD1 (Ab-206) Antibody in phosphorylation studies?

A robust experimental design for SMAD1 phosphorylation studies should include these essential controls:

Positive Controls:

• BMP-stimulated samples: Treat cells with BMP2 (25-100 ng/ml) for 1 hour to induce S206 phosphorylation. Research demonstrates that BMP treatment induces phosphorylation of the SMAD1 linker region and C-tail of SMAD1/5 .

• Constitutively active receptor expressing cells: Transfection with ALK3(Q233D) has been shown to increase P-SMAD1 levels and can serve as a reliable positive control .

• EGF-treated samples: Since S206 can also be phosphorylated by ERK in response to mitogenic growth factors, EGF treatment provides an alternative pathway for positive control .

Negative Controls:

• Phosphatase-treated lysates: Treatment with phosphatases like PPM1A has been shown to effectively reduce P-SMAD1 levels .

• CDK8/9 inhibition: RNAi knockdown of both CDK8 and CDK9 results in significant reduction of SMAD1-ALP, or alternatively, treatment with flavopiridol can be used as a pharmacological inhibitor .

• SMAD1 knockdown/knockout cells: Generate SMAD1-depleted cells using RNAi or CRISPR-Cas9 to validate antibody specificity.

Additional Methodology Controls:

• Total SMAD1 detection: Always run parallel samples with antibodies recognizing total SMAD1 (regardless of phosphorylation state) to normalize phosphorylation signals.

• Multiple detection methods: Validate findings using both immunoblotting and immunofluorescence to confirm localization patterns of phosphorylated SMAD1.

• Cross-reactivity assessment: Include SMAD5 and SMAD8 samples to verify antibody specificity, as these proteins share significant sequence homology with SMAD1.

How does the temporal dynamics of SMAD1 S206 phosphorylation compare with other phosphorylation events in the pathway?

The phosphorylation of SMAD1 follows a precise temporal sequence that regulates both signal activation and termination:

• Sequential Phosphorylation Cascade:

  • Initial event: C-terminal SXS motif phosphorylation by BMP receptor kinases

  • Secondary event: Linker region phosphorylation (including S206) by CDK8/9

  • Tertiary event: Additional linker phosphorylation by GSK3 at sites like T202 and S210

• Temporal Relationship Data:

  Phosphorylation Event	Typical Timing After BMP Stimulation	Functional Outcome	Reference
  C-terminal SXS motif	5-15 minutes	SMAD1 activation and nuclear translocation
  S206 phosphorylation	15-30 minutes	Transcriptional complex assembly and activation
  GSK3-mediated phosphorylation	30-60 minutes	Preparation for ubiquitination and degradation

• Differential Stability: Research has shown that flavopiridol (a CDK8/9 inhibitor) extends the half-life of BMP-activated SMAD1/5 as much as the proteasome inhibitor MG132, suggesting that linker phosphorylation, including at S206, initiates the protein degradation pathway .

• Subcellular Localization Dynamics: The phosphorylation at S206 occurs predominantly in the nucleus, as evidenced by studies showing that flavopiridol prevents ALP of nuclear SMAD1 in BMP-treated cells . This indicates that this modification happens after SMAD1 has been activated and translocated to the nucleus.

• Connection to Transcriptional Activity: ChIP experiments have demonstrated that phosphorylated SMAD1 (detectable by phospho-Ser206 antibody) is present on BMP responsive regions of target genes like Id1 and Smad7 . Notably, RNAP II inhibitor α-amanitin did not affect SMAD1 ALP, indicating that this phosphorylation accompanies, but is not a consequence of active transcription .

What mechanisms regulate the balance between SMAD1 S206 phosphorylation and dephosphorylation?

The phosphorylation status of SMAD1 at S206 is regulated through a complex interplay of kinases, phosphatases, and contextual cellular signals:

• Kinase Regulation:

  • CDK8/9 are the primary kinases that phosphorylate S206 in response to BMP signaling

  • ERK can also phosphorylate S206 in response to mitogenic growth factors or in cancer cells with activated Ras

  • RNAi inhibition studies have shown that knockdown of both CDK8 and CDK9 results in greater reduction of SMAD1-ALP than single knockdowns, suggesting these kinases act redundantly

• Phosphatase Activity:

  • While SCP4 specifically dephosphorylates the SXS motif of SMAD1 but not the S206 site , other phosphatases like PPM1A appear to have broader activity against phosphorylated SMAD1

  • The presence of site-specific phosphatases creates a sophisticated system for differential regulation of various phosphorylation sites

• Pathway Cross-regulation:

  • BMP-induced SMAD1 linker phosphorylation was not suppressed by inhibitors of MEK (U0126), p38 (SB203580), or JNK (SP600125), indicating pathway specificity

  • Of all protein kinase inhibitors screened, only flavopiridol effectively inhibited SMAD-ALP, confirming the specific role of CDK8/9

• Feedback Regulation:

  • The phosphorylated linker marks SMAD1 for ubiquitination by E3 ligases like SMURF1 and SMURF2

  • This creates an intrinsic negative feedback loop where activation (via phosphorylation) initiates the process leading to signal termination (via degradation)

• Context-dependent Regulation:

  • In cancer contexts like MT1-MMP expressing tumor cells, SMAD1 expression correlates with MT1-MMP levels, suggesting additional regulatory mechanisms in pathological states

  • Active TGF-β (but not latent TGF-β) can induce SMAD1 expression and 3D cell proliferation in MT1-MMP suppressed cells, demonstrating cross-talk between signaling pathways

How can researchers distinguish between specific and non-specific signals when interpreting SMAD1 (Ab-206) Antibody results?

Accurate interpretation of SMAD1 (Ab-206) Antibody data requires careful consideration of several factors:

• Signal Validation Approaches:

  • Molecular weight verification: Phosphorylated SMAD1 should appear at approximately 60 kDa on Western blots

  • Phosphatase treatment control: Compare untreated samples with phosphatase-treated samples; the specific signal should disappear after phosphatase treatment

  • SMAD1 knockdown/knockout: Signal should be absent or significantly reduced in cells lacking SMAD1

• Distinguishing Features of Authentic Signals:

  • Stimulus-dependent appearance: Signal should increase after BMP stimulation or expression of constitutively active ALK3(Q233D)

  • Inhibitor sensitivity: Signal should decrease with CDK8/9 inhibitors like flavopiridol

  • Subcellular localization: Phosphorylated SMAD1 should predominantly localize to the nucleus after BMP stimulation

• Potential Cross-reactivity Considerations:

  • SMAD family members: Due to sequence homology, verify that the signal isn't coming from other SMAD proteins, particularly SMAD5 or SMAD8

  • Other phosphorylated proteins: Check whether the signal pattern changes under conditions that shouldn't affect SMAD1 phosphorylation

• Quantitative Analysis Guidelines:

  • Normalization strategy: Always normalize phospho-SMAD1 signals to total SMAD1 levels to account for variations in protein expression

  • Signal linearity range: Perform dilution series to establish the linear detection range of the antibody

  • Statistical robustness: Perform sufficient biological replicates (minimum n=3) to account for inherent variability in phosphorylation levels

• Data Reconciliation Methods:

  • Multi-antibody approach: Compare results with antibodies recognizing different phosphorylation sites on SMAD1

  • Functional correlation: Verify that changes in phosphorylation correlate with expected functional outcomes, such as transcription of SMAD1 target genes like Id1

  • Orthogonal techniques: Validate important findings using mass spectrometry-based phospho-proteomics to confirm site-specific modifications

How can SMAD1 (Ab-206) Antibody be used to investigate the role of phosphorylated SMAD1 in embryonic stem cell differentiation?

SMAD1 (Ab-206) Antibody offers valuable insights into the molecular mechanisms governing stem cell fate decisions:

• Neural Differentiation Inhibition Analysis: Research has established that YAP recruitment to phosphorylated linker sites (including pS206) supports SMAD1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells . The antibody can be used to track this phosphorylation during differentiation protocols.

• Temporal Mapping Protocol: Follow these steps to map SMAD1 phosphorylation during differentiation:

  • Collect embryonic stem cells at defined time points during differentiation

  • Analyze pS206 levels by Western blotting and immunofluorescence

  • Correlate with expression of pluripotency markers (Oct4, Nanog) and lineage markers

  • Relate changes to BMP pathway activation status

• Genetic Manipulation Approach: Combine antibody detection with:

  • CRISPR-engineered SMAD1 phospho-site mutants (S206A)

  • CDK8/9 knockdown/inhibition

  • YAP modulation (overexpression, knockdown, or nuclear localization mutants)

• Single-cell Resolution Studies: Use flow cytometry or immunofluorescence with the SMAD1 (Ab-206) Antibody to:

  • Identify heterogeneity in BMP signaling within stem cell populations

  • Track correlation between pS206 levels and differentiation markers at the single-cell level

  • Monitor dynamic changes during embryoid body formation

• Target Gene Expression Correlation: The antibody can be used in ChIP experiments to track SMAD1 binding to regulatory regions of differentiation-associated genes. Studies have shown that antibodies against phospho-Ser206 of SMAD1 successfully pulled down BMP responsive regions of genes like Id1 , which is known to regulate stem cell differentiation.

What insights can SMAD1 (Ab-206) Antibody provide about the structural dynamics of WW domain interactions with phosphorylated SMAD1?

The antibody can be used to explore the complex structural dynamics of protein-protein interactions involving phosphorylated SMAD1:

• Co-immunoprecipitation Studies: Use the antibody to pull down pS206-SMAD1 and associated proteins, particularly WW domain-containing partners like YAP. Research has shown that the phosphorylated S206 and P207 side chains are accommodated in the aromatic cavity formed by Tyr188 and Trp199 in the YAP WW1 domain .

• In vitro Binding Analysis: Compare binding affinities between:

  Protein Variants	Binding Partner	Observed Affinity	Reference
  Unphosphorylated SMAD1	YAP WW1-WW2	Low affinity
  pS206 SMAD1	YAP WW1-WW2	Enhanced affinity
  pS206/pS214 SMAD1	YAP WW1-WW2	Further enhanced

• Structural Perturbation Analysis: Use the antibody to verify the effects of point mutations in:

  • The SMAD1 phosphorylation sites (S206A, P207A)

  • The WW domain aromatic pocket (Y188A, W199A in YAP)

  • The surrounding residues that stabilize the interaction

• Competitive Binding Assays: The antibody can help determine whether:

  • Different WW domain proteins compete for binding to pS206-SMAD1

  • Binding to pS206 affects accessibility of other phosphorylation sites

  • Sequential binding occurs among different WW domain proteins

• Real-time Binding Kinetics: Use surface plasmon resonance or biolayer interferometry with the antibody to:

  • Measure on/off rates for different WW domain proteins binding to pS206-SMAD1

  • Determine how phosphorylation at multiple sites affects binding dynamics

  • Assess how other modifications influence the interaction

How might SMAD1 (Ab-206) Antibody be utilized to explore the role of SMAD1 phosphorylation in pathological conditions?

The antibody provides a valuable tool for investigating SMAD1's involvement in disease processes:

• Cancer Signaling Pathway Analysis:

  • MT1-MMP (a key integral membrane protease) expressing tumor cells show correlation between MT1-MMP and SMAD1 expression

  • RNAi knockdown of SMAD1 in MT1-MMP expressing HT1080 cells impaired tumor growth

  • The antibody can be used to examine whether altered S206 phosphorylation plays a role in this process

• BMP Signaling Dysregulation in Disease:

  • Apply the antibody in tissue microarray analysis to:

    • Compare pS206 levels between normal and diseased tissues

    • Correlate with clinical outcomes and disease progression

    • Identify potential biomarker applications

• Drug Response Monitoring:

  • Use the antibody to track changes in SMAD1 phosphorylation in response to:

    • CDK8/9 inhibitors (like flavopiridol)

    • TGF-β pathway modulators

    • ERK pathway inhibitors (which may affect S206 phosphorylation through cross-talk)

• Genetic Disease Models:

  • Apply the antibody in studies of conditions with known BMP/TGF-β pathway mutations:

    • Hereditary hemorrhagic telangiectasia

    • Juvenile polyposis syndrome

    • Pulmonary arterial hypertension

• Therapeutic Target Validation:

  • The dual role of S206 phosphorylation in both activating transcription and marking SMAD1 for degradation makes it a potential therapeutic target

  • The antibody can help determine whether modulating this phosphorylation would have beneficial effects in specific pathological contexts

  • Monitor the effects of experimental therapeutics on SMAD1 phosphorylation status and downstream gene expression

What emerging technologies could enhance the utility of SMAD1 (Ab-206) Antibody in research?

Advanced technological approaches can significantly expand the research applications of this antibody:

• Proximity Ligation Assays (PLA): Combining the SMAD1 (Ab-206) Antibody with antibodies against potential interaction partners (like YAP or SMURF1) in PLA would allow visualization of specific protein-protein interactions involving phosphorylated SMAD1 at single-molecule resolution within cells.

• CRISPR-based Endogenous Tagging: Generating cell lines with endogenously tagged SMAD1 would allow correlation between antibody-detected phosphorylation and live-cell imaging of SMAD1 dynamics. This could reveal how S206 phosphorylation affects nuclear-cytoplasmic shuttling and protein stability in real time.

• Mass Spectrometry Integration: Using the antibody for immunoprecipitation followed by mass spectrometry could identify novel interaction partners specifically recruited to pS206-SMAD1, potentially revealing new components of the signaling pathway.

• Spatial Transcriptomics Correlation: Combining immunofluorescence using the antibody with spatial transcriptomics could reveal how the spatial distribution of pS206-SMAD1 correlates with expression patterns of target genes at single-cell resolution within tissues.

• Microfluidics-based Signaling Dynamics: Using the antibody in microfluidic systems allowing precise temporal control of BMP stimulation could provide insights into the kinetics of S206 phosphorylation and its relationship to other phosphorylation events in the pathway.

What are the most promising research questions that could be addressed using SMAD1 (Ab-206) Antibody?

The antibody enables investigation of several critical unresolved questions in SMAD1 biology:

• Phosphorylation Code Deciphering: How does the combination of multiple phosphorylation events (including S206) create a "code" that determines the fate and function of SMAD1? Research has shown that different phosphorylation patterns affect binding partner recruitment, with S206 phosphorylation facilitating YAP interaction .

• Transcriptional Burst Dynamics: Does S206 phosphorylation regulate the frequency or amplitude of transcriptional bursts from SMAD1 target genes? The antibody could be used in combination with MS2 tagging of target genes to correlate phosphorylation status with transcriptional dynamics.

• Developmental Timing Precision: How does the timing of S206 phosphorylation contribute to developmental decisions? The dual role in both activating transcription and marking SMAD1 for degradation suggests it may create precise temporal windows of pathway activity.

• Pathological Dysregulation Mechanisms: Is aberrant regulation of S206 phosphorylation a contributing factor in diseases with known BMP pathway involvement? The correlation between MT1-MMP and SMAD1 in tumor growth suggests potential pathological relevance.

• Evolutionary Conservation Analysis: How conserved is the regulatory mechanism involving S206 phosphorylation across species, and does it play similar roles in different developmental contexts? The antibody could be used to examine this site in various model organisms if cross-reactivity permits.

How might artificial intelligence and computational approaches enhance research using SMAD1 (Ab-206) Antibody data?

Computational methods can extract deeper insights from antibody-generated data:

• Phosphorylation Dynamics Modeling: Machine learning algorithms can integrate phospho-SMAD1 data with other pathway components to model the temporal dynamics of the BMP signaling network and predict system behavior under various perturbations.

• Image Analysis Automation: Deep learning-based image analysis can quantify subtle changes in pS206-SMAD1 levels, subcellular localization, and co-localization with other proteins across large datasets of immunofluorescence images.

• Multi-omic Data Integration: Computational approaches can correlate pS206-SMAD1 levels (detected by the antibody) with:

  • Transcriptomic data (RNA-seq of target genes)

  • Epigenomic data (ChIP-seq of SMAD1 binding sites)

  • Proteomic data (mass spectrometry of interaction partners)

• Structural Prediction Refinement: Data from antibody-based binding studies can inform molecular dynamics simulations of pS206-SMAD1 interactions with WW domain proteins, providing insights into binding mechanisms that are difficult to observe experimentally.

• Biomarker Pattern Recognition: AI algorithms can identify patterns in pS206-SMAD1 levels across patient samples that correlate with disease progression or treatment response, potentially leading to new diagnostic or prognostic approaches.

• Virtual Screening Applications: Computational drug discovery approaches can use structural insights from pS206-SMAD1 studies to identify small molecules that could selectively modulate this phosphorylation event or its downstream effects, opening new therapeutic possibilities.