Phospho-SMAD2 (S250) Antibody

Basic Research Questions

• What is the biological significance of SMAD2 phosphorylation at Serine 250?

  SMAD2 phosphorylation at Serine 250 occurs in the linker region, which is a regulatory domain between the MH1 and MH2 domains of SMAD2. This specific phosphorylation is part of the complex regulation of SMAD2 activity that modulates TGF-beta signaling. Unlike the C-terminal phosphorylation (at Ser465/467) which is directly catalyzed by the TGF-beta receptor and promotes SMAD2 activation, linker region phosphorylation at sites including Ser250 can regulate SMAD2 through various mechanisms including altering nuclear accumulation, affecting protein stability, and modulating interactions with transcriptional cofactors . Research indicates that phosphorylation at S250 may be particularly important for fine-tuning the transcriptional response to TGF-beta signaling in specific cellular contexts.

• How does SMAD2 S250 phosphorylation differ functionally from the canonical SMAD2 phosphorylation sites?

  The canonical SMAD2 phosphorylation occurs at C-terminal Ser465/467 residues in response to direct TGF-beta receptor activation. In contrast, S250 phosphorylation occurs in the linker region and is regulated by different kinases. While C-terminal phosphorylation primarily controls SMAD2 activation and nuclear translocation, S250 phosphorylation appears to modulate transcriptional activity after SMAD2 has been activated . Notably, S250 phosphorylation often occurs in conjunction with nearby sites (S245, S255) and potentially works in concert with these modifications to regulate SMAD2 function. The temporal dynamics also differ—C-terminal phosphorylation typically occurs rapidly after TGF-beta stimulation, whereas linker phosphorylation including S250 may follow different kinetics depending on the cellular context and upstream kinase activity.

• What experimental applications are most appropriate for Phospho-SMAD2 (S250) antibodies?

  Based on validated applications reported across multiple sources, Phospho-SMAD2 (S250) antibodies are suitable for several techniques:

  Application	Validated Success	Special Considerations
  Western Blot (WB)	High	1:500-1:5000 dilution range typical
  Flow Cytometry (FCM)	Good	Requires permeabilization
  Immunohistochemistry (IHC-P)	Moderate	Often requires antigen retrieval
  Immunofluorescence (IF)	Good	May need optimization for signal-to-noise ratio
  ELISA	Variable	Works with specific formats

  Western blotting remains the gold standard for detecting specific phosphorylation status, with a distinct band observed at approximately 60 kDa corresponding to phosphorylated SMAD2 .

• What cellular conditions or stimuli promote SMAD2 S250 phosphorylation?

  Several conditions have been documented to induce SMAD2 S250 phosphorylation:

  • TGF-β1 stimulation (100 pM) following serum starvation (16h) can induce phosphorylation of SMAD2 linker regions including S250

  • Activation of mitogenic signaling pathways, particularly through growth factor stimulation

  • Stress conditions, including cellular stress responses

  • Activity of specific kinases, particularly nemo-like kinase (NLK) has been documented to phosphorylate the SMAD2 linker region

  The timing of phosphorylation may vary, with some studies showing increased S250 phosphorylation within 30-60 minutes of stimulation, potentially followed by a slower dephosphorylation phase. Cell type-specific differences in phosphorylation kinetics have been observed across human cell lines including HaCaT keratinocytes, U2OS osteosarcoma cells, HeLa cells, and HEK293 cells .

Advanced Research Questions

• What are the specific kinases known to phosphorylate SMAD2 at S250, and how can their activity be experimentally manipulated?

  Several kinases have been implicated in SMAD2 S250 phosphorylation:

  • Nemo-like kinase (NLK) has been directly shown to phosphorylate the linker region of SMAD2, including at S250

  • Cyclin-dependent kinases (CDKs) may also contribute to linker region phosphorylation

  • ERK/MAPK pathway kinases have been implicated in SMAD linker phosphorylation

  To experimentally manipulate these kinases:

  1. Genetic approaches: Use CRISPR-based methods to generate kinase knockouts (e.g., NLK knockouts as described in source )

  2. Pharmacological inhibition: Apply specific kinase inhibitors at appropriate concentrations

  3. Point mutations: Generate kinase-dead mutants (e.g., NLK (K155M) or NLK (T286V)) to express dominant-negative forms

  4. RNAi approaches: Use siRNA transfection (20 nM final concentration with Lipofectamine RNAi MAX as demonstrated in )

  These approaches can be validated by monitoring SMAD2 S250 phosphorylation levels using the specific antibodies in Western blotting.

• How does phosphorylation at S250 affect SMAD2 complex formation with SMAD4 and subsequent transcriptional activity?

  Phosphorylation at S250 affects SMAD2-SMAD4 complex dynamics in several ways:

  1. Complex stability: S250 phosphorylation may alter the stability of the SMAD2-SMAD4 complex through conformational changes

  2. Nuclear retention: Evidence suggests that linker phosphorylation, including at S250, modulates the nuclear-cytoplasmic shuttling dynamics of SMAD2

  3. Transcriptional activity: Studies indicate that S250 phosphorylation can both potentiate or attenuate transcription depending on cellular context and the specific target genes

  Experimentally, the effect on transcription can be assessed through:

  • Chromatin immunoprecipitation (ChIP) assays using SMAD2/3 antibodies to detect binding to TGF-β responsive elements

  • Promoter-reporter assays that contain TGF-β responsive elements

  • Expression analysis of known TGF-β target genes such as PAI-1, P21, FN, CTGF, and P15

  When studying these interactions, it's important to consider that S250 phosphorylation often occurs alongside other linker region phosphorylations (S245, S255), requiring careful experimental design to isolate the specific contribution of S250.

• What are the recommended protocols for studying temporal dynamics of SMAD2 S250 phosphorylation?

  To effectively study the temporal dynamics of S250 phosphorylation:

  1. Time-course experiments:

     • Serum-starve cells for 16 hours to establish baseline

     • Stimulate with 100 pM TGF-β1 for various time points (0, 15, 30, 60, 120, 240 minutes)

     • Rapidly lyse cells in phosphatase inhibitor-containing buffer

  2. Pulse-chase analysis:

     • Stimulate with TGF-β, then add receptor kinase inhibitors to stop new phosphorylation

     • Monitor decay of S250 phosphorylation over time

  3. Live-cell imaging approaches:

     • Generate FRET-based biosensors incorporating the SMAD2 linker region

     • Monitor phosphorylation-induced conformational changes in real-time

  4. Multiplexed detection:

     • Simultaneously monitor S250 phosphorylation alongside canonical C-terminal phosphorylation (S465/467)

     • Compare with total SMAD2 levels and other phosphorylation sites

  In all cases, using phosphatase inhibitors during sample preparation is critical for preserving phosphorylation status. Additionally, parallel detection of total SMAD2 levels helps normalize for expression differences across samples.

• How can cross-reactivity between phospho-specific antibodies for different SMAD2 phosphorylation sites be detected and eliminated?

  To assess and prevent cross-reactivity between phospho-specific SMAD2 antibodies:

  1. Site-directed mutagenesis validation:

     • Generate SMAD2 constructs with specific serine-to-alanine mutations (e.g., S250A)

     • Express these mutants in SMAD2-knockout cells (available through CRISPR-Cas9 approaches)

     • Test antibody reactivity against wild-type vs. mutant proteins

  2. Peptide competition assays:

     • Pre-incubate antibodies with phosphorylated peptides containing the S250 site

     • Compare signal reduction with peptides containing other phosphorylation sites

  3. Phosphatase treatment controls:

     • Treat duplicate samples with lambda phosphatase prior to immunoblotting

     • Genuine phospho-specific signals should disappear after phosphatase treatment

  4. Mass spectrometry validation:

     • Perform immunoprecipitation with the phospho-specific antibody

     • Analyze the precipitated proteins by mass spectrometry to confirm the exact phosphorylation sites

     • Follow protocols similar to those described in , using LC-MS/MS analysis with Thermo Scientific Q Exactive mass spectrometer

  When multiple phosphorylation sites are in close proximity (e.g., S245/S250/S255), experimental caution is particularly important as some antibodies might recognize multiple phosphorylated residues in this region .

Methodological Considerations

• What sample preparation techniques ensure optimal detection of phospho-SMAD2 (S250) in Western blotting?

  For optimal detection of phospho-SMAD2 (S250):

  1. Cell lysis conditions:

     • Use ice-cold lysis buffer containing both phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) and protease inhibitors

     • Maintain samples at 4°C throughout processing

  2. Loading controls:

     • Always include total SMAD2 detection in parallel samples

     • Use housekeeping proteins (e.g., GAPDH) for normalization

  3. Gel separation:

     • Use 5-20% gradient SDS-PAGE gels for optimal separation

     • Run at 70V (stacking)/90V (resolving) for 2-3 hours

  4. Transfer conditions:

     • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

     • Verify transfer efficiency with reversible stains

  5. Blocking conditions:

     • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

     • For some phospho-epitopes, 5% BSA may provide better results than milk

  6. Antibody incubation:

     • Incubate with primary antibody at 0.5 μg/mL overnight at 4°C

     • Use antibody dilutions in the range of 1:500-1:5000 depending on antibody source

  Following these protocols should yield a distinct band at approximately 60 kDa corresponding to phosphorylated SMAD2 at S250 .

• What are the critical controls necessary when examining SMAD2 S250 phosphorylation in experimental systems?

  Essential controls include:

  1. Negative controls:

     • SMAD2 knockout cells generated by CRISPR-Cas9

     • Serum-starved cells without stimulation

     • Secondary antibody-only control

  2. Positive controls:

     • Cells treated with known inducers of SMAD2 phosphorylation (e.g., TGF-β1 at 100 pM)

     • Overexpression of active kinases known to phosphorylate S250

  3. Specificity controls:

     • Parallel detection with antibodies against other SMAD2 phosphorylation sites

     • SMAD2 mutants (S250A) expressed in knockout backgrounds

     • Peptide competition assays

  4. Normalization controls:

     • Total SMAD2 detection in parallel samples

     • Standard housekeeping proteins (GAPDH)

  5. Treatment validation:

     • Monitor canonical SMAD2 phosphorylation (Ser465/467) to confirm pathway activation

     • Functional readouts of pathway activity (e.g., target gene expression)

  These controls help distinguish specific phosphorylation events from background signals and ensure the biological relevance of observed changes in phosphorylation.

• What are the recommended approaches for quantifying relative levels of S250 phosphorylation?

  For accurate quantification of S250 phosphorylation:

  1. Western blot densitometry:

     • Capture images within the linear range of detection

     • Normalize phospho-SMAD2 (S250) signal to total SMAD2

     • Use software like ImageJ for density measurement

  2. Flow cytometry quantification:

     • Calculate median fluorescence intensity (MFI)

     • Compare MFI ratios between phospho-SMAD2 and total SMAD2

     • Generate histogram overlays to visualize shifts in phosphorylation

  3. ELISA-based approaches:

     • Consider sandwich ELISA formats that capture total SMAD2 and detect phospho-S250

     • Generate standard curves with recombinant phosphorylated proteins

  4. Multiplexed analysis:

     • Use systems that can simultaneously detect multiple phosphorylation sites

     • Consider phospho-flow cytometry for single-cell analysis

  5. Image analysis for IF/IHC:

     • Quantify nuclear versus cytoplasmic signal intensity

     • Use automated imaging systems with consistent exposure parameters

  In all approaches, biological and technical replicates are essential for statistical validity, and normalization to appropriate controls helps account for experimental variation.

• What are the best strategies for detecting phospho-SMAD2 (S250) in complex tissue samples?

  When working with complex tissues:

  1. Tissue preparation:

     • Fix tissues rapidly to preserve phosphorylation state

     • Consider using phosphatase inhibitors during processing

  2. Antigen retrieval optimization:

     • Test both citrate and EDTA-based retrieval methods

     • Optimize pH and heating conditions for specific tissues

  3. Signal amplification methods:

     • Consider tyramide signal amplification for low-abundance phosphorylation

     • Use polymer-based detection systems for enhanced sensitivity

  4. Multi-label approaches:

     • Co-stain for cell-type specific markers to identify expressing cells

     • Use sequential staining protocols for multiple phosphorylation sites

  5. Controls for tissue analysis:

     • Include phosphatase-treated sections as negative controls

     • Use tissues from pathway-activated and control conditions

     • Consider genetically modified tissue models (e.g., conditional knockouts)

  For human samples, proper tissue handling from collection to fixation is critical, as phosphorylation status can change rapidly post-mortem or during extended ischemia times.

• How can phospho-SMAD2 (S250) be effectively studied in co-culture or 3D culture systems?

  For advanced culture systems:

  1. Co-culture separation strategies:

     • Use cell-type specific markers for flow cytometry sorting

     • Consider using cells with fluorescent labels for identification

     • Employ microdissection techniques for physical separation

  2. In situ detection in 3D cultures:

     • Optimize fixation protocols to maintain structure while allowing antibody penetration

     • Use confocal microscopy with z-stack imaging for spatial resolution

     • Consider clearing techniques for deep imaging of larger organoids

  3. Pathway activation in complex systems:

     • Apply localized TGF-β stimulation using microfluidic devices

     • Use optogenetic approaches for spatiotemporal control of signaling

  4. Sample preparation from 3D cultures:

     • Develop specialized lysis protocols that maintain phosphorylation status

     • Consider cryosectioning followed by laser capture microdissection

  5. Single-cell analysis approaches:

     • Use phospho-flow cytometry to assess heterogeneity in phosphorylation

     • Consider single-cell Western technologies for complex samples

  These approaches help preserve the contextual information that is often critical for understanding phosphorylation dynamics in physiologically relevant systems.

• What are the key methodological differences when detecting phospho-SMAD2 (S250) across different model organisms?

  Important considerations across species:

  1. Cross-species reactivity verification:

     • Most phospho-SMAD2 (S250) antibodies recognize human, mouse, and rat SMAD2

     • Sequence alignment should be performed for other species

     • Validate antibodies in each species before experimental use

  2. Species-specific sample preparation:

     • Optimize lysis buffers for tissue-specific differences

     • Consider species variations in protease/phosphatase activity

  3. Fixation protocols across species:

     • Different tissues may require modified fixation times

     • Test multiple fixatives for optimal epitope preservation

  4. Pathway activation differences:

     • TGF-β concentration requirements may vary between species

     • Kinetics of phosphorylation/dephosphorylation can differ

  5. Control samples:

     • Generate species-matched positive controls

     • Consider using tissues from knockout animals when available

  The conservation of SMAD2 sequence across mammals makes many antibodies cross-reactive, but validation in each species remains essential for reliable results.

• How can mass spectrometry be used to complement antibody-based detection of SMAD2 S250 phosphorylation?

  Mass spectrometry offers powerful complementary approaches:

  1. Sample preparation for MS:

     • Immunoprecipitate SMAD2 using total SMAD2 antibodies

     • Digest with appropriate enzymes (trypsin and chymotrypsin are commonly used)

     • Enrich for phosphopeptides using TiO₂ or immobilized metal affinity chromatography

  2. LC-MS/MS analysis protocols:

     • Use nanoflow LC systems for optimal peptide separation

     • Consider using the Thermo Scientific Q Exactive mass spectrometer with 65-minute gradient elution at 0.30 μl/min

     • Include multiple fragmentation methods (HCD, ETD) for comprehensive analysis

  3. Data analysis approaches:

     • Use ptmRS node in Proteome Discoverer for phosphosite probability scoring

     • Consider site probabilities above 75% as confidently localized

     • Compare peptide fragmentation patterns with theoretical predictions

  4. Quantitative phosphoproteomics:

     • Use SILAC, TMT, or label-free quantification for relative abundance

     • Monitor multiple phosphorylation sites simultaneously

     • Include synthetic phosphopeptide standards for absolute quantification

  5. Integrated analysis:

     • Correlate MS-based quantification with antibody-based detection

     • Use MS to discover novel phosphorylation sites or patterns

     • Validate MS findings with site-specific antibodies

  Mass spectrometry provides unbiased detection of phosphorylation sites and can reveal complex patterns of multiple modifications that may not be detectable with antibody-based methods alone.