Phospho-SMAD2 (S250) Recombinant Monoclonal Antibody

What is the biological significance of SMAD2 phosphorylation at serine 250?

SMAD2 phosphorylation at serine 250 represents one of several key regulatory modifications within the TGF-β signaling pathway. SMAD2 functions as a receptor-regulated SMAD (R-SMAD) that acts as an intracellular signal transducer and transcriptional modulator activated by TGF-beta and activin type 1 receptor kinases . The phosphorylation at S250 occurs in the linker region of SMAD2, distinct from the canonical C-terminal phosphorylation at S465/467 that occurs directly through TGF-β receptor activation. Linker region phosphorylation, including at S250, represents an integration point for crosstalk between TGF-β and other signaling pathways, allowing for context-dependent modulation of SMAD2's transcriptional activities. This phosphorylation plays critical roles in regulating cell proliferation, differentiation, and apoptotic responses .

How does the structure and function of SMAD2 relate to its phosphorylation status?

SMAD2 belongs to the SMAD family of proteins, evolutionarily conserved from the Drosophila gene 'mothers against decapentaplegic' (Mad) and C. elegans gene Sma . Structurally, SMAD2 contains conserved MH1 and MH2 domains connected by a less conserved linker region where S250 resides. Upon TGF-β receptor activation, SMAD2 undergoes C-terminal phosphorylation, which induces dissociation from the SMAD anchor for receptor activation (SARA) protein and subsequent association with SMAD4 . This SMAD2/SMAD4 complex translocates to the nucleus, binds the TRE element in promoter regions of TGF-β-regulated genes, and activates transcription . The linker region phosphorylation at sites including S250 provides additional regulatory control, often mediated by non-TGF-β pathways, creating a sophisticated mechanism for signal integration and contextual responses to TGF-β.

What are the major cellular processes and pathways influenced by SMAD2 S250 phosphorylation?

SMAD2 S250 phosphorylation influences multiple cellular processes within the broader context of TGF-β signaling, including:

• Cell proliferation regulation: Phosphorylation at S250 can modulate the growth inhibitory effects of TGF-β signaling in various cell types

• Cellular differentiation: SMAD2 promotes TGF-β1-mediated transcription of differentiation genes, with S250 phosphorylation potentially regulating this activity

• Apoptotic responses: The phosphorylation status influences cell survival signals

• Cancer progression: SMAD2 may act as a tumor suppressor in colorectal carcinoma, with its phosphorylation status potentially affecting this function

• PDPK1 kinase activity: Phosphorylated SMAD2 positively regulates PDPK1 by stimulating its dissociation from the inhibitory 14-3-3 protein YWHAQ

What are the optimal conditions for detecting phospho-SMAD2 (S250) via Western blotting?

For optimal Western blot detection of phospho-SMAD2 (S250), researchers should follow these methodological guidelines:

• Sample preparation: Use whole cell lysates from appropriate cell lines (HeLa, HepG2, NIH/3T3, C6) with or without treatment with PMA (0.2μM for 0.5h) as a positive control

• Protein loading: Load 20-30μg of protein per lane for standard detection

• Blocking conditions: Use 5% non-fat dry milk in TBST as blocking and diluting buffer

• Primary antibody dilution: Apply the phospho-SMAD2 (S250) antibody at dilutions between 1:500-1:5000 depending on the specific product and sensitivity required

• Detection considerations: Be aware that phospho-SMAD2 (S250) typically appears as a band at approximately 60-65 kDa, though the calculated molecular weight is 52 kDa

• Potential degradation: Note that bands around and below the 50-kDa marker could represent degradation fragments

• Secondary antibody: Use an HRP-conjugated anti-rabbit secondary antibody at approximately 1:100,000 dilution

How can researchers validate the specificity of phospho-SMAD2 (S250) antibody detection?

Validating antibody specificity for phospho-SMAD2 (S250) is crucial for experimental reliability. A comprehensive validation approach should include:

• Peptide competition assays: Use phosphorylated and non-phosphorylated peptides to demonstrate binding specificity. Test against peptides containing different phosphorylation sites (S245, S250, S255) and combinations thereof to establish site-specificity

• Dot blot analysis: Apply a systematic dot blot with various phospho-peptides:

  • Smad2 (phospho S245+S250+S255) peptide

  • Smad2 (phospho S245+S250) peptide

  • Smad2 (phospho S245+S255) peptide

  • Smad2 (phospho S250+S255) peptide

  • Individual phosphorylation sites (S245, S250, S255)

  • Non-phosphorylated peptide as negative control

• Signal induction: Compare signals between untreated cells and cells treated with PMA (0.2μM for 0.5h), which induces phosphorylation

• Immunoprecipitation: Perform IP followed by Western blot to confirm specificity

• Knockout/knockdown controls: Include SMAD2 knockdown or knockout samples as negative controls

What immunoprecipitation protocols yield the best results with phospho-SMAD2 (S250) antibodies?

For optimal immunoprecipitation of phospho-SMAD2 (S250), follow these methodological guidelines:

• Sample preparation: Start with 0.35-0.5 mg of whole cell lysate from cells treated with phosphorylation inducers (e.g., 0.2μM PMA for 0.5h)

• Antibody amount: Use the phospho-SMAD2 (S250) antibody at approximately 1/30 dilution (about 2μg antibody per 0.35mg lysate)

• Immunoprecipitation procedure:

  • Incubate lysate with antibody overnight at 4°C

  • Add protein A/G beads and incubate for 2-4 hours

  • Wash beads thoroughly to remove non-specific binding

• Western blot detection: Perform Western blotting on immunoprecipitated material using the same phospho-SMAD2 (S250) antibody at 1/1000 dilution

• Secondary antibody: Use a specialized IP detection reagent (like VeriBlot for IP Detection Reagent) at 1/5000 dilution to avoid heavy and light chain interference

• Controls: Include a parallel IP with isotype control antibody (e.g., rabbit monoclonal IgG) to identify non-specific binding

How should researchers interpret discrepancies between calculated (52 kDa) and observed (60-65 kDa) molecular weights for phospho-SMAD2?

The discrepancy between the calculated molecular weight (52 kDa) and observed molecular weight (60-65 kDa) of phospho-SMAD2 is a common observation that requires careful interpretation:

• Post-translational modifications: Multiple phosphorylation sites beyond S250 (including canonical C-terminal sites S465/467 and other linker region sites) can contribute to reduced electrophoretic mobility, causing the protein to migrate more slowly than predicted

• Glycosylation or other modifications: Additional post-translational modifications may further alter migration patterns

• Isoform considerations: Alternative splicing variants of SMAD2 exist and may display different migration patterns

• Technical factors affecting migration:

  • Buffer composition and pH

  • Acrylamide percentage in SDS-PAGE gels

  • Running conditions

• Verification strategies:

  • Use total SMAD2 antibody alongside phospho-specific antibody to confirm identity

  • Include phosphatase-treated samples as controls to verify phosphorylation-dependent migration shift

  • Consider isoform-specific primers if investigating at mRNA level

The consistent observation of the 60-65 kDa band across multiple independent studies strongly suggests this represents the authentic phosphorylated form of SMAD2 .

What factors might contribute to inconsistent phospho-SMAD2 (S250) detection in experimental samples?

Inconsistent detection of phospho-SMAD2 (S250) can result from several experimental and biological factors:

• Cell type and context dependence:

  • Different cell lines may exhibit varying baseline and inducible S250 phosphorylation levels

  • Growth conditions and cell density can affect phosphorylation status

  • Cell cycle phase may influence phosphorylation patterns

• Sample processing issues:

  • Rapid phosphatase activity during lysate preparation can dephosphorylate target residues

  • Inadequate phosphatase inhibitors in lysis buffers

  • Protein degradation during sample preparation or storage

  • Freeze-thaw cycles degrading phospho-epitopes

• Technical considerations:

  • Antibody dilution optimization requirements

  • Blocking buffer compatibility issues (5% NFDM/TBST recommended)

  • Signal-to-noise ratio challenges with different detection systems

  • Batch-to-batch antibody variation

• Biological stimulus timing:

  • Transient nature of many phosphorylation events

  • Optimal time points after stimulus application may vary (0.5h PMA treatment used in validation studies)

• Troubleshooting approaches:

  • Include known positive controls (e.g., 293T+PMA, NIH/3T3+PMA)

  • Test multiple lysis buffer formulations with varying phosphatase inhibitor compositions

  • Optimize fixation methods for ICC/IF applications

  • Perform time-course experiments to identify peak phosphorylation

How can phospho-SMAD2 (S250) antibodies be utilized to investigate crosstalk between TGF-β and other signaling pathways?

Phospho-SMAD2 (S250) antibodies provide powerful tools for investigating signaling crosstalk through these methodological approaches:

• Dual pathway stimulation experiments:

  • Treat cells with TGF-β in combination with activators of other pathways (MAPK, PI3K/AKT, Wnt)

  • Analyze S250 phosphorylation status alongside canonical C-terminal phosphorylation (S465/467)

  • Compare temporal dynamics of different phosphorylation events

• Pathway inhibitor studies:

  • Apply specific inhibitors of non-TGF-β pathways and measure effects on S250 phosphorylation

  • Use pharmacological inhibitors in combination with genetic approaches (siRNA, CRISPR)

  • Create dose-response and time-course matrices to map pathway interactions

• Nuclear-cytoplasmic fractionation:

  • Given SMAD2's nucleocytoplasmic shuttling , compare subcellular distribution of phospho-S250 SMAD2

  • Correlate localization patterns with transcriptional outcomes

  • Apply techniques like proximity ligation assay to detect interactions between phospho-SMAD2 and other signaling components

• Chromatin immunoprecipitation (ChIP):

  • Use phospho-S250 antibodies for ChIP to identify genomic targets specifically regulated by this modification

  • Compare with targets of canonically phosphorylated SMAD2

  • Perform sequential ChIP to examine co-occupancy with other transcription factors

• Proteomics approaches:

  • Conduct immunoprecipitation with phospho-S250 antibodies followed by mass spectrometry

  • Identify differential interaction partners compared to non-phosphorylated or C-terminally phosphorylated SMAD2

  • Apply phosphoproteomics to map global signaling changes following pathway perturbations

What are the implications of studying S250 phosphorylation in cancer and developmental biology research?

Studying SMAD2 S250 phosphorylation has significant implications for both cancer and developmental biology research:

• Cancer research applications:

  • TGF-β pathway exhibits context-dependent tumor suppressor or promoter functions

  • SMAD2 may act as a tumor suppressor in colorectal carcinoma

  • S250 phosphorylation potentially serves as a biomarker for cancer progression or treatment response

  • Linker phosphorylation may mediate resistance to TGF-β-induced growth inhibition in cancer cells

  • Targeting enzymes responsible for S250 phosphorylation could represent therapeutic strategies

• Developmental biology implications:

  • SMAD2 promotes TGF-β1-mediated transcription of differentiation genes

  • S250 phosphorylation may regulate context-specific responses during embryonic development

  • The balance between different SMAD2 phosphorylation states may direct cell fate decisions

  • Temporal dynamics of phosphorylation could coordinate developmental timing

• Methodological approaches for both fields:

  • Tissue-specific expression analysis using immunohistochemistry with phospho-S250 antibodies

  • In vivo models with phosphorylation site mutations (S250A/S250E)

  • Single-cell analyses to examine heterogeneity in phosphorylation patterns

  • Correlation of phosphorylation status with differentiation markers or tumor progression indicators

How can researchers effectively study the dynamics of SMAD2 S250 phosphorylation in relation to other phosphorylation sites?

Studying the dynamic interplay between different SMAD2 phosphorylation sites requires sophisticated methodological approaches:

• Multiplex phosphorylation analysis:

  • Use antibodies specific for individual phosphorylation sites (S245, S250, S255, S465/467)

  • Apply multiplexed Western blotting with different fluorescent secondary antibodies

  • Develop ELISA-based assays to quantitatively measure multiple phosphorylation sites

  • Consider phospho-flow cytometry for single-cell resolution of multiple phosphorylation events

• Time-course experiments:

  • Apply stimulus (TGF-β, PMA, etc.) and collect samples at multiple time points

  • Compare temporal patterns of different phosphorylation events

  • Use mathematical modeling to infer causality between phosphorylation events

• Mutagenesis approaches:

  • Generate phospho-mimetic (S→D/E) and phospho-null (S→A) mutations at different sites

  • Create combination mutants to study interdependence of phosphorylation events

  • Analyze functional consequences of mutations on SMAD2 localization, protein interactions, and transcriptional activity

• Mass spectrometry-based approaches:

  • Apply phosphoproteomics to quantitatively assess multiple phosphorylation sites simultaneously

  • Use SILAC or TMT labeling for comparative analysis across conditions

  • Perform targeted mass spectrometry for enhanced sensitivity to specific phosphopeptides

• Advanced imaging techniques:

  • Develop and apply phosphorylation-specific FRET biosensors

  • Use phospho-specific antibodies for super-resolution microscopy

  • Apply live cell imaging to track phosphorylation dynamics in real-time

How does the detection of phospho-SMAD2 (S250) compare across different experimental techniques (WB, ELISA, IP, ICC/IF)?

Each experimental technique offers distinct advantages and limitations for phospho-SMAD2 (S250) detection:

What considerations should be made when selecting cell lines or tissue samples for phospho-SMAD2 (S250) studies?

When selecting experimental systems for phospho-SMAD2 (S250) studies, researchers should consider:

• Baseline expression and phosphorylation levels:

  • Human cell lines: HeLa and HepG2 cells are validated as suitable models

  • Rodent models: NIH/3T3 (mouse) and C6 (rat) cells show detectable levels after stimulation

  • Consider natural variation in SMAD2 expression across tissue types

• Response to stimulation:

  • PMA treatment (0.2μM for 0.5h) has been validated to induce S250 phosphorylation

  • Different cell types may require optimization of stimulus concentration and duration

  • Consider multiple stimuli (PMA, TGF-β, serum, stress inducers) for comprehensive analysis

• Experimental system characteristics:

  • TGF-β pathway status (receptor expression, pathway mutations)

  • Context of other relevant signaling pathways

  • Growth characteristics and culture requirements

  • Transfection/transduction efficiency if genetic manipulation is required

• Technical considerations:

  • Antibody cross-reactivity with species-specific variants (human, mouse, rat)

  • Extraction efficiency of phosphorylated proteins from different sample types

  • Sample availability and ethical considerations for tissue samples

• Disease relevance:

  • For cancer studies, consider cell lines that reflect the cancer type of interest

  • For developmental studies, consider models that recapitulate relevant differentiation processes

  • Patient-derived samples may provide greater clinical relevance but introduce heterogeneity

What are the key technical differences between various commercially available phospho-SMAD2 (S250) antibodies?

When selecting a phospho-SMAD2 (S250) antibody, researchers should consider these key technical parameters: