Phospho-SMAD1 (Ser187) Antibody

Basic Research Questions

• What is the significance of SMAD1 phosphorylation at Serine 187 in cellular signaling pathways?

  SMAD1 phosphorylation at Serine 187 represents a critical regulatory mechanism within the TGF-β superfamily signaling pathway. This phosphorylation event occurs in the linker region of SMAD1 and plays a pivotal role in modulating SMAD1's transcriptional activity. Phosphorylation at Ser187 is primarily mediated by MAPK enzymes (such as ERK, p38, and JNK) and affects the nuclear localization, stability, and activity of SMAD1 .

  This specific phosphorylation is particularly important because:

  • It can regulate BMP-induced SMAD1 signaling

  • It serves as a point of cross-talk between the TGF-β/BMP pathway and other signaling cascades

  • It affects the duration and strength of SMAD1-mediated transcriptional responses

  • It's subject to dephosphorylation by phosphatases like SCPs (Small C-terminal Domain Phosphatases)

• How does SMAD1 (Ser187) phosphorylation differ from C-terminal SXS phosphorylation in function and regulation?

  SMAD1 undergoes two distinct types of phosphorylation with different regulatory implications:

  Phosphorylation Type	Kinases Responsible	Cellular Localization	Functional Outcome
  C-terminal SXS motif	BMP receptor kinases	Initially cytoplasmic	Activation of SMAD1, promotes nuclear translocation and transcriptional activity
  Linker region (including Ser187)	MAPK enzymes (ERK, p38, JNK), GSK-3	Can occur in nucleus after initial SXS phosphorylation	Generally inhibitory, can cause nuclear exclusion, promote ubiquitination and degradation

  The sequential nature of these phosphorylation events is critical: BMP receptor first causes SMAD1 C-terminal phosphorylation and nuclear translocation; SMAD1 is then phosphorylated by MAPK enzymes in the nucleus at linker sites including Ser187; finally, GSK-3 can further phosphorylate the pre-phosphorylated linker region . This creates a sophisticated regulatory system that fine-tunes the strength and duration of BMP signaling.

Experimental Methods and Applications

• What are the optimal applications for Phospho-SMAD1 (Ser187) antibodies in TGF-β/BMP pathway research?

  Phospho-SMAD1 (Ser187) antibodies are versatile tools for investigating the regulation of TGF-β/BMP signaling. Based on the literature and product specifications, these antibodies can be effectively employed in:

  • Western Blot (WB): To detect and quantify phosphorylated SMAD1 at Ser187 in cell or tissue lysates. This is particularly useful for studying temporal changes in phosphorylation following stimulation with growth factors or other treatments .

  • Immunohistochemistry (IHC): For visualizing the tissue and cellular distribution of phosphorylated SMAD1 in paraffin-embedded or frozen tissue sections .

  • Immunofluorescence/Immunocytochemistry (IF/ICC): To examine subcellular localization of phospho-SMAD1 and potential co-localization with other proteins .

  • Cell-Based ELISA: For high-throughput quantitative measurement of phospho-SMAD1 levels in intact cells without the need for cell lysis .

  • Transcription Factor Activity Assays: To assess the functional impact of Ser187 phosphorylation on SMAD1's transcriptional activity .

• What are the critical parameters for successful Western blot detection of Phospho-SMAD1 (Ser187)?

  For optimal Western blot detection of Phospho-SMAD1 (Ser187), researchers should consider the following methodological parameters:

  • Sample Preparation: Rapid lysis of cells/tissues in the presence of phosphatase inhibitors is essential to preserve phosphorylation status. Standardized protein quantification is critical for comparative analysis.

  • Expected Molecular Weight: The anticipated band size for phospho-SMAD1 is approximately 60/55 kDa (observed) or 52 kDa (calculated) . Multiple bands may reflect different phosphorylation states or isoforms.

  • Controls: Include positive controls (cells treated with BMP or TGF-β), negative controls (phosphatase-treated lysates), and loading controls (typically GAPDH for whole-cell lysates).

  • Antibody Dilution: The optimal dilution should be determined experimentally, but manufacturers typically recommend starting with dilutions in the range of 1:500-1:2000 .

  • Signal Detection: Phospho-specific antibodies may require enhanced chemiluminescence with longer exposure times compared to total protein detection.

  • Quantification: For accurate phosphorylation analysis, normalize phospho-SMAD1 signal to total SMAD1 rather than just to loading controls.

Advanced Research Applications

• How can Phospho-SMAD1 (Ser187) antibodies be applied to study cross-talk between TGF-β/BMP and other signaling pathways?

  Phospho-SMAD1 (Ser187) antibodies serve as powerful tools for investigating signaling cross-talk because the linker phosphorylation represents an integration point between multiple pathways:

  • Pathway Interaction Studies: By using specific inhibitors of MAPK pathways (ERK, p38, JNK) alongside BMP or TGF-β stimulation, researchers can assess how different signaling cascades modulate SMAD1 Ser187 phosphorylation. For example, studies have shown that inhibitors of TGF-β/Smad signaling like SB525334 can reduce phospho-Smad1/5/9 immunoreactivity in astrocytes after ischemia .

  • Temporal Analysis: Time-course experiments combining phospho-SMAD1 (Ser187) detection with phosphorylation status of other signaling molecules can reveal the sequence of activation events and regulatory feedback loops.

  • Nuclear-Cytoplasmic Fractionation: Because MAPK-directed phosphorylation can affect nuclear exclusion of SMAD1, combining subcellular fractionation with phospho-SMAD1 detection provides insights into compartment-specific regulation .

  • Phosphatase Studies: Investigating the role of phosphatases like SCPs or PPM1A in modulating SMAD1 phosphorylation status offers insights into signal termination mechanisms. Research has demonstrated that SCPs can dephosphorylate Smad1 in the linker at Ser206, and presumably Ser187 and Ser195, in both mammalian cells and Xenopus embryos .

• What experimental approaches can reveal the functional consequences of SMAD1 Ser187 phosphorylation in developmental processes?

  To elucidate the functional impact of SMAD1 Ser187 phosphorylation in development, researchers can employ several sophisticated approaches:

  • Phospho-mimetic and Phospho-resistant Mutations: Generating SMAD1 constructs with S187D/E (phospho-mimetic) or S187A (phospho-resistant) mutations allows assessment of how constitutive phosphorylation or absence of phosphorylation affects SMAD1 function in developmental contexts.

  • In Vivo Developmental Models: Analyzing phospho-SMAD1 (Ser187) patterns in developmental systems like Xenopus embryos can reveal stage-specific and tissue-specific regulation. This is particularly relevant as SCPs have been shown to dephosphorylate Smad1 at both the C-terminus and linker regions in Xenopus, affecting secondary axis development .

  • Lineage-Specific Conditional Expression: Using tissue-specific promoters to express modified SMAD1 can reveal how Ser187 phosphorylation affects specific developmental lineages.

  • Quantitative Transcriptomics: RNA-seq analysis of cells expressing wild-type versus mutant SMAD1 can identify target genes specifically regulated by Ser187 phosphorylation status.

  • Chromatin Immunoprecipitation (ChIP): Using phospho-SMAD1 (Ser187) antibodies for ChIP experiments can identify genome-wide binding sites affected by this specific phosphorylation.

Troubleshooting and Data Interpretation

• How should researchers address potential cross-reactivity of Phospho-SMAD1 (Ser187) antibodies with other phosphorylated SMAD proteins?

  Cross-reactivity is a significant concern when working with phospho-specific antibodies. For Phospho-SMAD1 (Ser187) antibodies:

  • Sequence Homology Analysis: Examine the amino acid sequences surrounding Ser187 in SMAD1 and corresponding regions in other SMADs. High homology suggests potential cross-reactivity.

  • Validation Controls: Include lysates from cells expressing only one SMAD family member or use SMAD knockout cell lines as controls.

  • Phosphatase Treatment: Treating half of your sample with lambda phosphatase should eliminate the specific band if it genuinely represents phosphorylated protein.

  • Immunodepletion: Sequential immunoprecipitation with antibodies against different SMAD proteins can help identify the specific contribution of each SMAD to the observed signal.

  • Peptide Competition: Using phosphorylated and non-phosphorylated peptides corresponding to the Ser187 region of SMAD1 can confirm antibody specificity.

  The sequence conservation among SMAD family members necessitates these validation steps, particularly when studying systems where multiple SMADs are expressed and activated simultaneously.

• What methods can be used to analyze the temporal dynamics of SMAD1 Ser187 phosphorylation following BMP or TGF-β stimulation?

  To effectively capture the dynamic nature of SMAD1 Ser187 phosphorylation:

  • Time-Course Western Blotting: Collect samples at multiple time points (typically 0, 5, 15, 30, 60, 120, 240 minutes, and 24 hours) after stimulation to generate phosphorylation kinetic profiles.

  • Cell-Based ELISA: Enables high-throughput analysis of phosphorylation dynamics in intact cells. The SMAD1 Phospho-Ser187 Colorimetric Cell-Based ELISA Kit can detect phosphorylated SMAD1 expression in as few as 5,000 cells .

  • Live-Cell Imaging: Using fluorescent biosensors based on phospho-specific antibody fragments or FRET-based reporters can provide real-time visualization of phosphorylation events.

  • Phospho-Proteomics: Mass spectrometry-based approaches can quantify changes in multiple phosphorylation sites simultaneously, providing a comprehensive view of SMAD1 modification status.

  • Mathematical Modeling: Integrating experimental data into computational models can help predict and understand the complex interplay between different phosphorylation events and their functional outcomes.

  These approaches are particularly valuable for understanding how linker phosphorylation at Ser187 relates temporally to C-terminal phosphorylation and subsequent functional changes in SMAD1 activity.

Pathological Contexts and Therapeutic Implications

• How is SMAD1 Ser187 phosphorylation altered in pathological conditions, and what methodologies best capture these changes?

  SMAD1 Ser187 phosphorylation undergoes significant alterations in various pathological contexts:

  • Cerebral Ischemia: Research using rat models of global cerebral ischemia has demonstrated increased phosphorylated Smad1/5/9-immunopositive astrocytes in the CA1 region 7 days after ischemia. This phosphorylation appears to be mediated by TGF-β1, as intracerebroventricular injection of SB525334, an inhibitor of TGF-β/Smad signaling, reduced phospho-Smad1/5/9 immunoreactivity .

  • Methodological Approaches for Pathological Assessment:

    Method	Advantages	Best Applications
    Immunohistochemistry	Preserves tissue architecture, allows cellular localization	Tissue biopsies, animal models
    Western blotting	Quantitative, allows comparison between samples	Cell lines, tissue lysates
    Phospho-flow cytometry	Single-cell resolution, multiple parameters	Blood samples, dissociated tissues
    Cell-based ELISA	High-throughput, no lysate preparation	Cultured primary cells, cell lines

  • Dual Staining Approaches: Combining phospho-SMAD1 detection with markers of pathological processes (e.g., GFAP for astrogliosis) provides contextual information about which cell types exhibit altered phosphorylation in disease states.

• What are the latest research findings regarding the role of SMAD1 Ser187 phosphorylation in modulating therapeutic responses?

  Recent research has highlighted several important aspects of SMAD1 Ser187 phosphorylation in therapeutic contexts:

  • Neural Protection and Repair: Studies in rat models of global cerebral ischemia suggest that TGF-β1-induced Smad1/5/9 phosphorylation in astrocytes may play a role in post-ischemic events, including brain inflammation and tissue repair, though it appears not to be directly involved in neuroprotection .

  • Phosphatase Targeting: The identification of specific phosphatases like SCPs that dephosphorylate SMAD1 at both the C-terminus and linker regions presents potential therapeutic targets. Modulating these phosphatases could differentially affect TGF-β and BMP signaling pathways simultaneously .

  • Pathway Cross-talk: Understanding how SMAD1 Ser187 phosphorylation integrates signals from multiple pathways is crucial for predicting responses to targeted therapies. For instance, inhibitors targeting MAPK pathways may indirectly affect BMP signaling through altered SMAD1 linker phosphorylation.

  • Biomarker Potential: Changes in phospho-SMAD1 patterns could serve as biomarkers for disease progression or treatment response, particularly in contexts where TGF-β/BMP signaling plays a significant role.

  • Experimental Approaches: Transcription factor activity assays specifically designed for Phospho-SMAD1 (Ser187) enable researchers to directly measure how therapeutic interventions affect SMAD1's functional activity, rather than just its phosphorylation status .

Technical Considerations for Special Applications

• What are the optimal cell fixation and permeabilization conditions for immunocytochemical detection of Phospho-SMAD1 (Ser187)?

  For optimal immunocytochemical detection of phosphorylated proteins like Phospho-SMAD1 (Ser187), proper fixation and permeabilization are critical:

  • Fixation Protocol:

    • For adherent cells: 4% paraformaldehyde for 15 minutes at room temperature is generally effective

    • For suspension cells: 8% formaldehyde is recommended for cell fixation

  • Permeabilization Options:

    • 0.1-0.5% Triton X-100 in PBS for 10 minutes

    • 100% ice-cold methanol for 10 minutes (may better preserve phospho-epitopes)

    • 0.5% saponin can be gentler for certain applications

  • Critical Considerations:

    • Rapid fixation after stimulation is essential to preserve phosphorylation status

    • Include phosphatase inhibitors in all buffers prior to fixation

    • Optimize blocking conditions to reduce background (typically 5-10% serum corresponding to secondary antibody species)

    • Allow adequate incubation time with primary antibody (overnight at 4°C is often optimal)

  • Cell Density: For cell-based assays, the cells should be around 75-90% confluent. For HeLa cells, for example, 30,000 cells per well is recommended for overnight seeding .

• How can researchers effectively use Phospho-SMAD1 (Ser187) antibodies to study the spatial regulation of BMP signaling in tissue sections?

  Studying spatial regulation of BMP signaling in tissues requires specialized approaches:

  • Tissue Preparation:

    • For frozen sections: Quick fixation in 4% PFA followed by cryoprotection

    • For paraffin sections: Use antigen retrieval methods (typically citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Dual/Multiple Immunolabeling Strategies:

    • Combine Phospho-SMAD1 (Ser187) with total SMAD1 antibodies to assess the proportion of phosphorylated protein

    • Use cell-type-specific markers to identify responding cell populations

    • Include BMP receptor antibodies to correlate receptor expression with downstream signaling

  • Advanced Imaging Techniques:

    • Confocal microscopy for precise subcellular localization

    • Tissue clearing methods (CLARITY, CUBIC, etc.) for 3D visualization in thick specimens

    • Quantitative image analysis to measure phospho-SMAD1 intensity across tissue regions

  • Controls and Validation:

    • Include tissues from BMP-deficient or receptor knockout models

    • Treat control tissues with phosphatases to verify specificity

    • Compare with phospho-SMAD1/5/9 C-terminal antibodies to distinguish linker from C-terminal phosphorylation patterns

  These approaches have been successfully applied in studies examining phospho-Smad1/5/9 in rat brain following ischemic injury, revealing specific upregulation in astrocytes in affected regions .