Phospho-SMAD3 (S213) Antibody

What is the biological significance of SMAD3 phosphorylation at Serine 213?

Phosphorylation of SMAD3 at Serine 213 (S213) represents a critical regulatory mechanism within the TGF-β signaling pathway. Unlike the C-terminal phosphorylation (Ser423/425) that is directly mediated by TGF-β Receptor I, S213 phosphorylation occurs in the linker region and follows a different temporal pattern. This phosphorylation appears to regulate SMAD3's transcriptional activity and its ability to form complexes with other transcriptional modulators.

Research indicates that S213 phosphorylation occurs later than C-terminal phosphorylation, with evidence showing that while C-terminal phosphorylation peaks at approximately 30 minutes after TGF-β treatment, linker region phosphorylation (including S213) peaks at approximately 1 hour post-stimulation . This sequential phosphorylation suggests a regulatory mechanism where C-terminal phosphorylation may be a prerequisite for subsequent linker region modifications .

How do I distinguish between phosphorylation at S213 and other SMAD3 phosphorylation sites in my experiments?

To specifically detect SMAD3 phosphorylation at S213, researchers should employ phospho-site-specific antibodies that have been validated for S213 recognition. Several commercial antibodies are specifically designed to detect SMAD3 phosphorylated only at S213, including polyclonal antibodies generated using KLH-conjugated synthetic phosphopeptides corresponding to amino acid residues surrounding S213 of human SMAD3 .

When designing experiments to distinguish between different phosphorylation sites:

• Always include appropriate controls, including non-phosphorylated SMAD3 controls and samples treated with phosphatase

• Consider using multiple antibodies that target different phosphorylation sites (e.g., S213, S204, and Ser423/425) in parallel experiments

• Validate antibody specificity using SMAD3 knockout cell lines as negative controls

• Confirm findings using complementary techniques such as mass spectrometry

A time-course analysis can also help distinguish S213 phosphorylation from C-terminal phosphorylation, as they follow different kinetics after TGF-β stimulation .

What is the optimal protocol for detecting Phospho-SMAD3 (S213) in Western blotting applications?

For optimal detection of Phospho-SMAD3 (S213) in Western blotting applications, the following methodology is recommended:

Sample Preparation:

• Treat cells with TGF-β (typically 5 ng/ml) for approximately 1 hour to maximize S213 phosphorylation

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

• Normalize protein concentration prior to gel loading

Western Blotting Protocol:

• Separate proteins on SDS-PAGE (approximately 52 kDa for SMAD3)

• Transfer to nitrocellulose or PVDF membrane

• Block with 5% non-fat dry milk in TBST

• Incubate with anti-Phospho-SMAD3 (S213) antibody at 1:1000 dilution

• Wash and incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence

Expected Results:

• A phosphorylated band at approximately 52 kDa representing phospho-SMAD3 (S213)

• Intensity typically peaks at 1 hour post-TGF-β treatment

For verification of results, parallel blots with antibodies against total SMAD3 and other phosphorylation sites (e.g., Ser423/425) are recommended to establish the specificity and relationship between different phosphorylation events.

How does the kinetics of S213 phosphorylation compare with other SMAD3 phosphorylation sites?

Research demonstrates distinct temporal patterns of phosphorylation at different SMAD3 sites following TGF-β stimulation:

These distinct phosphorylation kinetics suggest a sequential regulatory mechanism where C-terminal phosphorylation precedes and may be necessary for the subsequent linker region phosphorylation events. Evidence indicates that while the TGF-β receptor is necessary for linker phosphorylation, the receptor itself does not directly phosphorylate these sites, pointing to a more complex signaling cascade involving intermediate kinases .

This temporal separation may represent a mechanism for fine-tuning the duration and specificity of SMAD3-mediated transcriptional responses to TGF-β stimulation.

What experimental conditions are optimal for inducing and detecting S213 phosphorylation in cultured cells?

For optimal induction and detection of SMAD3 S213 phosphorylation in cell culture systems:

Stimulation Protocol:

• Use purified, recombinant TGF-β at 5 ng/ml concentration

• Establish a time course (0, 5, 15, 30, 60, 120 minutes)

• Serum-starve cells (0.1% FBS) for 4-6 hours prior to stimulation to reduce background phosphorylation

• Maintain cells at 37°C with 5% CO₂ during stimulation

Cell Types:

• Epithelial cell lines such as Mv1Lu, HaCaT, and HT-1080 show robust responses

• Human cancer cell lines including HeLa can also be used effectively

Detection Methods:

• Western blotting (as described in Q3)

• Immunohistochemistry at 1:50-1:100 dilution

• Immunofluorescence at 1:50 dilution

Critical Considerations:

• Include appropriate controls (untreated cells, phosphatase-treated samples)

• Consider using kinase inhibitors to probe the signaling pathway (e.g., GSK3 inhibitors like LiCl can help distinguish phosphorylation mechanisms)

• For immunohistochemistry applications, formalin-fixed and paraffin-embedded tissues have been successfully used with these antibodies

What are the known kinases responsible for S213 phosphorylation and how can they be experimentally manipulated?

While the search results don't explicitly identify the kinase responsible for S213 phosphorylation, they do provide information about related phosphorylation sites that can guide experimental approaches:

Known Information:

• GSK3 has been identified as responsible for Ser204 phosphorylation in the SMAD3 linker region

• The TGF-β receptor does not directly phosphorylate linker sites including S213, despite being necessary for this phosphorylation

• C-tail phosphorylation by TGF-β Receptor I appears to be a prerequisite for linker region phosphorylation

Experimental Approaches to Identify the S213 Kinase:

• Chemical inhibitor screening:

  • Test various kinase inhibitors (MAPKs, CDKs, GSK3) to identify which prevent S213 phosphorylation

  • Use lithium chloride (LiCl) as a GSK3 inhibitor control (shown to inhibit Ser204 but not other sites)

• Kinase knockdown/knockout studies:

  • Systematically knock down candidate kinases using siRNA or CRISPR-Cas9

  • Monitor effects on S213 phosphorylation after TGF-β stimulation

• In vitro kinase assays:

  • Test purified kinases for ability to phosphorylate SMAD3 at S213 in vitro

  • Use recombinant SMAD3 proteins with mutations at C-terminal sites to test dependency

• Phosphorylation site mutations:

  • Generate S213A mutants to confirm antibody specificity

  • Create combinations of phosphorylation site mutations to test interdependence

How does phosphorylation at S213 affect SMAD3's interaction with other transcriptional regulators?

SMAD3 functions as a critical transcriptional modulator that, upon TGF-β stimulation, can form various protein complexes to regulate gene expression. Phosphorylation at S213 may modulate these interactions in several ways:

Known SMAD3 Interactions:

• SMAD3/SMAD4 complexes form to activate transcription of TGF-β regulated genes

• SMAD3/SMAD4/JUN/FOS complexes can form at AP-1/SMAD sites

• Phosphorylation status affects nuclear translocation and DNA binding

Experimental Approaches to Study S213's Role in Protein Interactions:

• Co-immunoprecipitation studies comparing wild-type SMAD3 vs. S213A mutants

• Chromatin immunoprecipitation (ChIP) using phospho-specific antibodies to map genomic binding sites

• Proximity ligation assays to visualize protein interactions in situ

• Transcriptional reporter assays with TGF-β responsive elements

For ChIP applications with phospho-SMAD3 antibodies, researchers should use approximately 5 μl of antibody with 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation for optimal results .

What are the technical challenges in generating and validating phospho-specific antibodies for SMAD3 S213?

Generating and validating highly specific phospho-SMAD3 (S213) antibodies presents several technical challenges:

Antibody Generation Challenges:

• Ensuring specificity for the phosphorylated S213 epitope while avoiding cross-reactivity with non-phosphorylated SMAD3 or other phosphorylation sites

• Existing antibodies are typically generated using KLH-conjugated synthetic phosphopeptides corresponding to amino acid residues surrounding S213

• Polyclonal antibodies may exhibit batch-to-batch variation, requiring rigorous validation of each lot

Validation Approaches:

• Western blot analysis comparing:

  • TGF-β stimulated vs. unstimulated cells

  • Phosphatase-treated vs. untreated lysates

  • Wild-type SMAD3 vs. S213A mutant expression

• Specificity confirmation using:

  • SMAD3 knockout cell lines as negative controls

  • Peptide competition assays with phosphorylated vs. non-phosphorylated peptides

  • Cross-validation with multiple antibodies from different sources

• Application-specific validation:

  • Western blotting (1:1000 dilution)

  • Immunohistochemistry (1:50-1:100 dilution)

  • Immunofluorescence (1:50 dilution)

  • ELISA

The published validation data demonstrates that these antibodies can detect endogenous levels of SMAD3 when phosphorylated at S213 in multiple applications .

How do dysregulated patterns of SMAD3 S213 phosphorylation contribute to pathological conditions?

While the search results don't provide explicit information about pathological conditions specifically linked to S213 phosphorylation, they do mention several disease contexts where SMAD3 dysregulation is important:

SMAD3-Associated Pathologies:

• Fibrosis (>967 publications linking SMAD3 to fibrosis)

• Inflammation (>373 publications)

• Cancer (multiple references to cancer tissues in immunohistochemistry studies)

• Pediatric T-cell lymphoblastic leukemia (associated with loss of SMAD3)

• Scleroderma (potentially related to SMAD3 upregulation)

Research Approaches to Study S213 Phosphorylation in Disease:

• Compare S213 phosphorylation patterns in normal vs. diseased tissues using immunohistochemistry

• Analyze the effects of S213A mutations on disease models in vitro and in vivo

• Correlate S213 phosphorylation levels with disease progression and treatment response

• Investigate how therapeutic interventions targeting TGF-β signaling affect S213 phosphorylation

For immunohistochemistry applications, formalin-fixed and paraffin-embedded human cancer tissues (including breast carcinoma and hepatocarcinoma) have been successfully used with anti-phospho-SMAD3 (S213) antibodies .

How can I design experiments to investigate the crosstalk between S213 phosphorylation and other post-translational modifications of SMAD3?

Investigating the interplay between different post-translational modifications of SMAD3 requires carefully designed experiments:

Experimental Strategies:

• Sequential immunoprecipitation:

  • First immunoprecipitate with anti-phospho-S213 antibody

  • Then probe the immunoprecipitate with antibodies against other modifications (e.g., phospho-Ser423/425, ubiquitination, acetylation)

• Site-directed mutagenesis:

  • Generate single and combination mutations (S213A, S423/425A)

  • Assess how each mutation affects other modifications

  • Evaluate functional consequences using reporter assays

• Temporal analysis:

  • Perform detailed time-course studies after TGF-β stimulation

  • Monitor multiple modifications simultaneously

  • Existing data shows C-terminal phosphorylation (30 min peak) precedes linker phosphorylation (60 min peak)

• Kinase/phosphatase manipulations:

  • Use specific inhibitors (e.g., TGF-β receptor inhibitors, GSK3 inhibitors)

  • Assess effects on different phosphorylation sites

  • Evidence suggests C-tail phosphorylation by TGF-β receptor is necessary for linker phosphorylation

• Mass spectrometry:

  • Perform phospho-proteomics to identify all modifications simultaneously

  • Quantify relative abundances of different modification patterns