Phospho-SMAD1 (Ser206) Antibody

What is Phospho-SMAD1 (Ser206) Antibody and what specific epitope does it recognize?

Phospho-SMAD1 (Ser206) Antibody is a research tool designed to specifically detect SMAD1 protein only when phosphorylated at the serine 206 residue in the linker region. This antibody recognizes a specific phosphorylated epitope (typically P-H-S(p)-P-T) derived from human SMAD1 . Unlike antibodies targeting the C-terminal phosphorylation sites (Ser463/465), this antibody provides insight into linker region phosphorylation, which is critical for understanding the inhibitory regulation of SMAD1 .

The antibody is typically produced by immunizing rabbits with synthetic phosphopeptides corresponding to the region containing serine 206, followed by affinity purification to ensure specificity for the phosphorylated form of the protein . Importantly, non-phospho-specific antibodies are removed during purification to minimize background and cross-reactivity with the non-phosphorylated form of SMAD1 .

How does SMAD1 Ser206 phosphorylation differ functionally from C-terminal phosphorylation?

The phosphorylation of SMAD1 occurs at distinct sites with opposing functional consequences:

Unlike C-terminal phosphorylation which activates SMAD1, linker phosphorylation at Ser206 inhibits SMAD1 activity through cytoplasmic retention and degradation . Interestingly, the linker region of SMAD1 can be simultaneously phosphorylated at Ser206 with or without BMP-4 stimulation, whereas C-terminal phosphorylation is strictly BMP-dependent . This dual phosphorylation system creates a sophisticated regulatory mechanism balancing activation and inhibition of SMAD1-mediated transcription .

Which species and cell types are suitable for investigation with Phospho-SMAD1 (Ser206) Antibody?

Based on the product specifications and experimental validations, Phospho-SMAD1 (Ser206) antibodies show confirmed reactivity with:

The antibody has been successfully used in various cell types including C2C12 mouse myoblasts, HaCaT keratinocytes, NIH/3T3 fibroblasts, and embryonic tissues . When using this antibody with species not directly tested, researchers should perform validation experiments as sequence homology alone (even 100% homology) does not guarantee reactivity .

What are the optimal applications and dilution protocols for Phospho-SMAD1 (Ser206) Antibody?

The Phospho-SMAD1 (Ser206) Antibody has been validated for multiple applications with specific recommended dilutions:

For Western blotting, a 1:500 dilution in 1% blocking buffer with incubation for 1 hour at room temperature has been successfully employed . Signal development typically requires ECL detection systems, and the expected molecular weight of phosphorylated SMAD1 is approximately 60 kDa . For optimal results, researchers should include appropriate positive controls (e.g., BMP4-stimulated cells) and negative controls (e.g., phosphatase-treated samples) .

How should samples be prepared to preserve and detect SMAD1 Ser206 phosphorylation?

Sample preparation is critical for preserving phosphorylation states:

• Cell Stimulation Protocols:

  • For positive controls: Treat cells with BMP-4 (50 ng/mL) for 30 minutes

  • For MAPK pathway activation: Treat with EGF for 1 hour

  • For TGF-β pathway: Treat with TGF-β1 for 1 hour

  • For hyperphosphorylation: Pre-treat with Calyculin A (100 ng/ml, a phosphatase inhibitor) for 15 minutes before BMP stimulation

• Lysis Conditions:

  • Use phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers

  • Include protease inhibitors to prevent degradation

  • Maintain cold temperatures throughout processing

  • Perform rapid lysis to minimize phosphatase activity

• Experimental Timing:

  • The linker region phosphorylation at Ser206 can be detected both with and without BMP-4 stimulation

  • TGF-β-induced Smad1 phosphorylation peaks later (30-75 min) than BMP-induced phosphorylation (15 min)

For optimal detection, samples should be processed quickly and kept cold to minimize dephosphorylation by endogenous phosphatases .

What controls and validation methods ensure specific detection of phosphorylated SMAD1 at Ser206?

To ensure specificity and reliability:

• Positive Controls:

  • BMP-4 treated cells (increases both C-terminal and linker phosphorylation)

  • Calyculin A-treated cells (phosphatase inhibitor enhances phosphorylation signals)

  • Constitutively active ALK3(Q233D) expressing cells

• Negative Controls:

  • Untreated/serum-starved cells

  • Phosphatase (e.g., SCP1, PPM1A) overexpressing cells

  • Dorsomorphin-treated cells (inhibits BMP receptor kinases)

  • Lambda phosphatase-treated lysates

• Specificity Validation:

  • Peptide competition assays using phosphorylated and non-phosphorylated peptides

  • Knockdown/knockout of SMAD1 (ensures band corresponds to SMAD1)

  • Parallel detection with total SMAD1 antibody

  • Phosphorylation site mutants (S206A mutant should show no signal)

• Cross-Reactivity Assessment:

  • Test for reactivity with SMAD5/SMAD8 (close homologs)

  • Western blot band pattern analysis (single band at 60 kDa is expected)

The antibody should detect a single band at approximately 60 kDa that increases with appropriate stimulation and decreases with phosphatase treatment or inhibition of upstream kinases .

How can Phospho-SMAD1 (Ser206) Antibody be used to study crosstalk between TGF-β and BMP signaling pathways?

Phospho-SMAD1 (Ser206) Antibody offers a unique window into pathway crosstalk:

• Dual Pathway Activation Experiments:

  • Research has revealed that TGF-β can significantly induce Smad1 phosphorylation in non-endothelial cell lineages

  • Using Phospho-SMAD1 (Ser206) Antibody alongside C-terminal phosphorylation antibodies can reveal differential regulation patterns

• Temporal Dynamics Analysis:

  • TGF-β-induced Smad1 phosphorylation peaks later (30-75 min) and is more transient than BMP-induced phosphorylation (peaks at 15 min)

  • Time-course experiments with both pathways can reveal sequential phosphorylation events

• Receptor Specificity Studies:

  • ALK5 (TGF-β type I receptor) can phosphorylate Smad1, challenging the traditional pathway separation

  • Using receptor-specific inhibitors (e.g., Dorsomorphin for ALK2/3/6) alongside Phospho-SMAD1 (Ser206) detection helps delineate which receptors mediate linker phosphorylation

• Cell-Type Specific Responses:

  • TGF-β-induced Smad1 phosphorylation has been observed in C2C12, HepG2, MEFs, and HaCaT cells

  • Comparative analysis across cell types can reveal tissue-specific pathway integration

This approach helps resolve the complex interplay between BMP and TGF-β signaling, which has significant implications for understanding development, disease, and potential therapeutic interventions .

What is the relationship between SMAD1 Ser206 phosphorylation and transcriptional regulation?

The phosphorylation of SMAD1 at Ser206 has profound effects on transcriptional activity:

• Phosphorylation-Dependent Protein Interactions:

  • Phosphorylation by CDK8/9 creates binding sites for YAP1, a transcriptional co-activator

  • Subsequent phosphorylation by GSK3 switches off YAP1 binding and adds binding sites for SMURF1 (E3 ubiquitin ligase)

• Nuclear Localization and Retention:

  • Linker phosphorylation at Ser206 inhibits nuclear accumulation of SMAD1 in response to BMP stimulation

  • In embryonic tissues, both linker-phosphorylated and tail-phosphorylated SMAD1 show nuclear localization with high co-localization in specific developmental contexts

• Transcriptional Target Regulation:

  • Phosphorylation at Ser206 affects BMP-responsive element (BRE) activity

  • SCP1 suppression of BMP-Smad signaling axis-induced osteoblastic differentiation involves Runx2

• Temporal Control of Signaling:

  • The dual phosphorylation system (activation at C-terminus, inhibition at linker region) creates a sophisticated time-dependent regulatory mechanism

  • This allows for transient activation followed by signal termination to ensure proper developmental outcomes

Researchers can leverage Phospho-SMAD1 (Ser206) Antibody alongside chromatin immunoprecipitation (ChIP) to investigate how this phosphorylation affects genomic targeting and transcriptional activities in different cellular contexts .

How do phosphatases regulate SMAD1 Ser206 phosphorylation in different cellular contexts?

Several phosphatases play crucial roles in modulating SMAD1 phosphorylation:

Research findings indicate that:

• The expression of wild-type SCP1, but not mutant SCP1, reduces BMP-4-induced phosphorylation of SMAD1 at both the C-terminus and linker region (Ser206) .

• Despite their overlapping functions, knockdown of PPM1A fails to completely restore phospho-SMAD1 levels, suggesting additional phosphatases are involved in SMAD1 regulation .

• Phosphatase activity is spatiotemporally regulated:

  • Dephosphorylation by PPM1A induces export from the nucleus to the cytoplasm

  • This dephosphorylation is inhibited by association with EGR1

  • SCP4 specifically targets SMAD1 but not all Smad proteins (PPM1D and SCP1 showed no detectable effects on SMAD1 phosphorylation)

• The balance between kinase and phosphatase activities determines the duration and intensity of SMAD1 signaling, with implications for cell fate decisions in development and disease contexts.

These findings highlight the complex regulation of SMAD1 phosphorylation by multiple phosphatases with overlapping yet distinct functions .

What are common technical challenges when using Phospho-SMAD1 (Ser206) Antibody and how can they be addressed?

Researchers commonly encounter several technical challenges:

• Low Signal Intensity:

  • Cause: Rapid dephosphorylation during sample preparation

  • Solution: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers; consider pre-treatment with Calyculin A

• High Background:

  • Cause: Insufficient blocking or cross-reactivity

  • Solution: Optimize blocking conditions (5% non-fat dry milk in TBST is effective) ; increase antibody dilution; use phosphopeptide competition

• Multiple Bands:

  • Cause: Cross-reactivity with other SMAD proteins or degradation products

  • Solution: Validate with SMAD1 knockdown; compare with total SMAD1 antibody; ensure sample integrity with protease inhibitors

• Inconsistent Results:

  • Cause: Variability in cell stimulation or phosphorylation status

  • Solution: Standardize stimulation protocols; include positive controls (BMP4-treated cells) in each experiment; normalize to total SMAD1 or housekeeping proteins

• Species Cross-Reactivity Issues:

  • Cause: Differences in epitope conservation across species

  • Solution: Despite predicted cross-reactivity based on sequence homology, validate experimentally for each new species

For reliable detection, always include both positive (BMP-stimulated) and negative (unstimulated or phosphatase-treated) controls in parallel with experimental samples .

How can researchers quantitatively analyze SMAD1 Ser206 phosphorylation across different experimental conditions?

Quantitative analysis requires systematic approaches:

• Western Blot Quantification:

  • Normalize phospho-SMAD1 (Ser206) signal to total SMAD1 protein

  • Use digital image analysis software to measure band intensity

  • Include a standard curve with known quantities of phosphorylated protein

  • Present data as fold-change relative to control conditions

• ELISA-Based Detection:

  • HTRF (Homogeneous Time Resolved Fluorescence) cell-based assays provide precise quantification

  • MSD phosphoprotein assays offer rapid quantification in small-volume samples

  • The PhosphoTracer ELISA approach can simultaneously measure phospho and total SMAD1

• Cell-Based Assays:

  • For multi-sample analysis, use 96-well format with dual detection systems

  • Calculate normalized results by dividing phospho-SMAD1 fluorescence by total GAPDH fluorescence in each well

  • Generate dose-response curves for treatments (as shown with BMP-4 concentrations from 0-30 ng/mL)

• Microscopy-Based Quantification:

  • Immunofluorescence with phospho-specific antibodies can reveal subcellular localization

  • Analyze co-localization with other signaling components

  • Quantify nuclear/cytoplasmic ratios to assess functional outcomes

For comparative analysis across experiments, include internal reference standards and present data as fold-change relative to standardized controls to account for inter-experimental variability .

How can researchers distinguish between SMAD1, SMAD5, and SMAD8 phosphorylation when using phospho-specific antibodies?

Distinguishing between closely related SMAD proteins requires careful experimental design:

• Antibody Selection Strategy:

  • Some phospho-antibodies are specific to SMAD1 (particularly for Ser206), while others detect multiple SMADs (e.g., Phospho-Smad1/5/8 antibodies for C-terminal sites)

  • The phospho-SMAD1 (Ser206) antibody has been reported not to cross-react with SMAD5 in some studies

• Validation Approaches:

  • Immunoprecipitation with SMAD-specific antibodies followed by phospho-detection

  • siRNA/shRNA knockdown of individual SMADs to confirm band identity

  • Overexpression of tagged versions of each SMAD

  • Use of SMAD knockout cell lines

• Technical Considerations:

  • C-terminal phosphorylation sites are highly conserved between SMAD1/5/8, making specific detection challenging

  • Linker regions show greater sequence divergence, offering better specificity

  • Molecular weight differences are subtle (SMAD1: 52-60 kDa)

• Analytical Approaches:

  • High-resolution SDS-PAGE can sometimes separate the closely related proteins

  • Mass spectrometry-based approaches can definitively identify specific phosphorylated peptides

  • Combining multiple antibodies (phospho-specific and SMAD-specific) can help resolve ambiguities

When absolute specificity is required, researchers should consider using CRISPR/Cas9-generated SMAD knockout cell lines to confirm the identity of detected bands or employing mass spectrometry-based phosphoproteomics for definitive identification .

What are the key considerations when comparing results from different phospho-SMAD1 antibodies in the same experiment?

When using multiple phospho-SMAD1 antibodies targeting different sites:

• Epitope Differences and Accessibility:

  • Antibodies targeting different phosphorylation sites (e.g., Ser206 vs. Ser463/465) may have different affinities and epitope accessibility

  • Structural changes induced by one phosphorylation event might affect detection of other sites

  • Some epitopes may be masked by protein-protein interactions

• Temporal Dynamics Considerations:

  • C-terminal phosphorylation (Ser463/465) is rapidly induced by BMP stimulation

  • Linker phosphorylation (Ser206) can occur both with and without BMP stimulation

  • TGF-β-induced Smad1 phosphorylation peaks later (30-75 min) than BMP-induced phosphorylation (15 min)

• Pathway-Specific Effects:

  • BMP pathway primarily induces C-terminal phosphorylation

  • MAPK pathway primarily affects linker region phosphorylation

  • TGF-β can induce both depending on context and timing

• Standardization Approaches:

  • Run parallel samples for each antibody rather than stripping and reprobing

  • Include the same positive and negative controls for all antibodies

  • Normalize to total SMAD1 for each sample

  • Consider developing a standard curve with known quantities of phosphorylated protein

By systematically comparing results from antibodies targeting different phosphorylation sites, researchers can gain insights into the sequential and potentially interdependent nature of these modifications and their functional consequences .

How can Phospho-SMAD1 (Ser206) Antibody be used to study developmental processes and disease models?

The regulatory role of SMAD1 phosphorylation makes it valuable for studying various biological contexts:

• Developmental Biology Applications:

  • In E13.5 mouse embryos, phospho-linker SMAD1 and phospho-tail SMAD1/5 show nuclear localization with high co-localization in specific tissues

  • Detection in ventricular zones of brain ventricles, tooth buds, spinal cord canal, and dorsal root ganglia

  • Moderate levels in gastric wall, developing heart valves, and epithelial cells of lung bronchioles and kidney tubules

• Bone and Cartilage Development:

  • SCP1 inhibits osteoblastic differentiation induced by the BMP-Smad axis via Runx2

  • Defects in Smad1 signaling cause bone-related disorders such as osteoporosis

  • BMP signaling through SMAD1 modulates expression of downstream osteogenic genes

• Cancer Research Applications:

  • SMAD1 is involved in metastatic progression of many cancer types

  • Can be induced by tumor-stimulating cytokines like BMP2 and TNFα

  • Plays important roles in cell invasion and metastasis

  • TGF-β/BMP pathway alterations are common in carcinogenesis

• Stem Cell Studies:

  • SMAD1 regulates transcription of genes critical to stem cell renewal

  • Phosphorylation status affects differentiation potential

  • BMP/SMAD1 signaling influences lineage decisions

This antibody provides a powerful tool for investigating how linker phosphorylation contributes to developmental patterning, disease progression, and cellular differentiation across diverse biological systems .

What methodological approaches can integrate phospho-SMAD1 detection with other signaling pathway components?

Comprehensive signaling analysis requires multi-dimensional approaches:

• Multiplexed Phosphoprotein Detection:

  • Combine phospho-SMAD1 (Ser206) detection with antibodies targeting:

    • C-terminal phosphorylated SMAD1 (Ser463/465)

    • Phosphorylated MAPK pathway components (ERK, p38, JNK)

    • Phosphorylated GSK3β (modulates SMAD1 signaling)

    • Activated BMP and TGF-β receptors

  • Use multiplexed ELISA or bead-based systems for quantitative multi-analyte detection

• Sequential Phosphorylation Analysis:

  • CDK8/9 phosphorylation creates binding sites for YAP1

  • Subsequent GSK3 phosphorylation switches off YAP1 binding and adds binding sites for SMURF1

  • Combining phospho-specific antibodies with protein interaction studies can reveal these sequential events

• Spatial Analysis Techniques:

  • Co-immunofluorescence to examine subcellular localization

  • Proximity ligation assays to detect protein-protein interactions dependent on phosphorylation

  • ChIP-Seq to examine genome-wide binding patterns influenced by phosphorylation status

• Systems Biology Approaches:

  • Combine phospho-SMAD1 detection with transcriptomics to link signaling to gene expression changes

  • Phosphoproteomics to identify global phosphorylation changes

  • Mathematical modeling to predict pathway dynamics based on experimental data

These integrated approaches provide a systems-level understanding of how SMAD1 phosphorylation coordinates with other signaling events to control cellular responses in development and disease .