Phospho-SMAD2 (S255) Recombinant Monoclonal Antibody

What is the SMAD2 protein and its role in TGF-β signaling?

SMAD2 is a receptor-regulated SMAD (R-SMAD) that functions as an intracellular signal transducer and transcriptional modulator activated by transforming growth factor-beta (TGF-β) and activin type 1 receptor kinases. It binds to the TRE element in the promoter region of many genes regulated by TGF-β and, upon forming a complex with SMAD4, activates transcription of target genes. SMAD2 plays a critical role in promoting TGF-β1-mediated transcription of various differentiation genes, including those involved in odontoblastic differentiation in dental papilla cells. Additionally, SMAD2 positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ, which acts as a negative regulator. Some research suggests SMAD2 may function as a tumor suppressor in colorectal carcinoma .

What is the significance of SMAD2 phosphorylation at the S255 residue?

Phosphorylation at S255 in the linker region of SMAD2 represents a key regulatory mechanism for modulating TGF-β signaling dynamics. This specific phosphorylation site is part of a cluster (including S245, S250, and S255) that regulates SMAD2 stability, nuclear accumulation, and transcriptional activity. Phosphorylation at S255 affects SMAD2's interaction with other regulatory proteins and influences its degradation rate, thereby controlling the duration and intensity of TGF-β signal transduction. Studies have shown that phosphorylation in the linker region often counteracts the canonical C-terminal phosphorylation in response to TGF-β signals, providing a mechanism for fine-tuning cellular responses to TGF-β stimulation .

How are Phospho-SMAD2 (S255) recombinant monoclonal antibodies produced?

Phospho-SMAD2 (S255) recombinant monoclonal antibodies are developed using protein technology and DNA recombinant techniques through a multi-step process:

• Antibody gene acquisition: An animal is immunized with a synthesized peptide derived from human phospho-SMAD2 (S255), followed by isolation of B cells.

• Screening process: The isolated B cells undergo screening to identify positive clones, followed by single clone identification.

• Genetic amplification: The light and heavy chains of the phospho-SMAD2 (S255) antibody are amplified via PCR.

• Vector construction: The amplified chains are integrated into a plasmid vector to construct a recombinant vector.

• Host cell transfection: The recombinant vector is transfected into host cells to facilitate antibody expression.

• Purification: The antibody is purified from cell culture supernatant using affinity chromatography.

• Validation: Stringent validation is conducted to ensure accuracy and efficacy for detecting human SMAD2 protein phosphorylated at the S255 residue in applications such as ELISA and immunofluorescence (IF) .

What are the optimal applications for Phospho-SMAD2 (S255) recombinant monoclonal antibodies?

Phospho-SMAD2 (S255) recombinant monoclonal antibodies are primarily optimized for immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA) applications. For IF applications, the recommended dilution range is 1:20-1:200, allowing researchers to visualize the subcellular localization and expression patterns of phosphorylated SMAD2 in fixed cells or tissue sections. These antibodies are particularly valuable for studying dynamic changes in SMAD2 phosphorylation in response to TGF-β pathway activation, inhibition, or modulation by various kinases such as NLK. The high specificity of recombinant monoclonal antibodies ensures reliable detection of the phosphorylated S255 residue specifically, minimizing cross-reactivity with other phosphorylation sites or related proteins .

How can phosphorylation sites on SMAD2 be accurately identified and validated in experimental settings?

Accurate identification and validation of SMAD2 phosphorylation sites require a combination of techniques:

• Mass Spectrometry Analysis: LC-MS/MS analysis provides the most definitive identification of phosphorylation sites. Purified GST-tagged SMAD2 proteins are incubated with the kinase of interest, separated by SDS-PAGE, and gel bands are excised for in-gel digestion with trypsin and chymotrypsin. Peptides are then analyzed using high-resolution mass spectrometry.

• Site-Directed Mutagenesis: Potential phosphorylation sites identified by mass spectrometry can be confirmed by substituting serine/threonine (S/T) residues with alanine/valine (A/V) to prevent phosphorylation. These mutant proteins are then subjected to in vitro kinase assays to verify the impact of the mutation.

• Phospho-specific Antibodies: Antibodies specifically recognizing phosphorylated residues (e.g., pSer255) can be raised and used in various assays (Western blot, ELISA, IF) to detect site-specific phosphorylation.

• Functional Assays: The physiological relevance of specific phosphorylation sites can be evaluated by expressing wild-type, phosphorylation-diminishing (S/A), or phosphorylation-mimicking (S/D) SMAD2 mutants in cells and assessing their effects on protein stability, transcriptional activity, and TGF-β signaling .

What controls should be included when using Phospho-SMAD2 (S255) antibodies in experimental workflows?

A robust experimental design using Phospho-SMAD2 (S255) antibodies should include the following controls:

How does NLK-mediated phosphorylation of SMAD2 differ from canonical TGF-β receptor-mediated phosphorylation?

Nemo-like kinase (NLK) and TGF-β receptor-mediated phosphorylation of SMAD2 represent distinct regulatory mechanisms that differentially impact TGF-β signaling:

NLK-mediated phosphorylation:

• Targets the linker region of SMAD2, particularly at sites analogous to S208 in SMAD3 (including S250 in SMAD2)

• Counteracts canonical C-terminal phosphorylation induced by TGF-β receptors

• Decreases the stability of SMAD2 protein, promoting its degradation

• Inhibits TGF-β-mediated transcriptional and cellular responses

• Functions as a negative feedback mechanism to control signal duration

Canonical TGF-β receptor-mediated phosphorylation:

• Targets the C-terminus of SMAD2

• Promotes SMAD2 nuclear accumulation and transcriptional activity

• Initiates TGF-β signal transduction

• Activates target gene expression through formation of SMAD2/SMAD4 complexes

This dual phosphorylation mechanism creates a molecular switch that allows precise temporal control of TGF-β signaling. Depletion of NLK enhances C-terminal phosphorylation of SMAD2 and increases SMAD2 protein levels, intensifying TGF-β responses. Conversely, overexpression of NLK enhances linker region phosphorylation while inhibiting C-terminal phosphorylation, attenuating TGF-β signaling .

What is the impact of SMAD2 linker region phosphorylation on protein stability and TGF-β signaling dynamics?

The linker region phosphorylation of SMAD2 serves as a critical regulatory mechanism for protein stability and TGF-β signaling:

• Protein Turnover Regulation:

  • Phosphorylation at sites like S250 in the linker region accelerates SMAD2 protein turnover

  • Phosphomimetic mutations (S/D) significantly decrease protein stability compared to wild-type SMAD2

  • Phosphorylation-resistant mutations (S/A) enhance protein stability, resulting in slower degradation

• Signal Duration Control:

  • Linker phosphorylation serves as a timer for TGF-β signaling by promoting SMAD2 degradation

  • This creates a negative feedback loop that helps terminate the signal after appropriate duration

  • Without proper linker phosphorylation, TGF-β signaling may persist inappropriately

• Transcriptional Activity Modulation:

  • Linker phosphorylation by NLK inhibits TGF-β-induced transcriptional activation

  • When linker phosphorylation is prevented by S/A mutations, the inhibitory effect of NLK on TGF-β signaling is impaired

  • This results in higher levels of transcriptional activation in response to TGF-β stimulation

Experimental evidence using wild-type, S/A, and S/D SMAD2/3 variants in cells with endogenous SMAD2/3 depletion has demonstrated that the fastest protein turnover occurs with phosphomimetic mutations, followed by wild-type proteins, with phosphorylation-resistant mutants showing the slowest degradation. This sophisticated regulatory mechanism ensures appropriate duration and intensity of TGF-β signaling in various cellular contexts .

How can mass spectrometry be optimized for identification of novel SMAD2 phosphorylation sites?

Optimizing mass spectrometry for novel SMAD2 phosphorylation site identification requires a comprehensive technical approach:

• Sample Preparation:

  • Express and purify GST-tagged SMAD2 proteins from bacteria for in vitro kinase assays

  • Include both untreated controls and samples incubated with potential kinases

  • Perform reduction with DTT (5 mM) and alkylation with iodoacetamide (11 mM)

  • Conduct parallel digestions with multiple proteases (trypsin and chymotrypsin) to improve sequence coverage

• LC-MS/MS Parameters:

  • Use a high-resolution mass spectrometer (e.g., Q Exactive) for accurate mass determination

  • Implement a gradient elution (65 min) with a flow rate of 0.30 μl/min

  • Utilize C-18 analytical columns (75 μm ID, 150 mm length) for optimal peptide separation

  • Set mobile phase composition: A (0.1% formic acid), B (80% acetonitrile, 0.1% formic acid)

  • Configure data-dependent acquisition mode with full-scan (350-1800 m/z, 70,000 resolution)

  • Apply normalized collision energy of 29% for HCD fragmentation

• Data Analysis:

  • Search spectra against SMAD2 protein database using search engines like Proteome Discoverer

  • Configure search parameters: no enzyme specificity, two missed cleavages allowed

  • Set carbamidomethylation (C) as fixed modification; oxidation (M) and phosphorylation as variable modifications

  • Apply stringent mass tolerances: 20 ppm for precursor ions, 0.02 Da for fragment ions

  • Use ptmRS node for phosphorylation site localization and probability assessment

  • Consider sites with ptmRS probabilities above 75% as truly modified

• Validation Strategies:

  • Confirm mass spectrometry findings with site-directed mutagenesis

  • Test phosphorylation of mutant proteins in vitro

  • Develop phospho-specific antibodies for detected sites

  • Evaluate the physiological relevance of identified sites through functional assays

This comprehensive approach has successfully identified multiple phosphorylation sites in SMAD2/3, including those targeted by NLK, providing valuable insights into the regulation of TGF-β signaling .

What are common challenges in detecting phospho-SMAD2 (S255) and how can they be addressed?

Researchers frequently encounter several challenges when detecting phospho-SMAD2 (S255) in experimental settings. These challenges and their solutions include:

• Low Signal Intensity:

  • Cause: Insufficient phosphorylation levels or antibody concentration

  • Solution: Optimize stimulation conditions (duration, concentration of stimulus); use recommended antibody dilutions (1:20-1:200 for IF); implement signal amplification techniques; increase protein loading for Western blots

• High Background:

  • Cause: Non-specific binding, excessive antibody concentration

  • Solution: Optimize blocking conditions; titrate antibody concentration; increase washing steps; use phospho-blocking reagents; pre-absorb antibody with non-phosphorylated peptide

• Cross-reactivity:

  • Cause: Antibody recognizing other phosphorylation sites or related proteins

  • Solution: Validate specificity using phospho-deficient mutants (S255A); perform peptide competition assays; use knockout controls; compare results with alternative antibodies

• Rapid Dephosphorylation:

  • Cause: Phosphatase activity during sample preparation

  • Solution: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers; maintain samples at 4°C; minimize processing time

• Inconsistent Results:

  • Cause: Variations in cell culture conditions, sample handling, or antibody lots

  • Solution: Standardize experimental protocols; include positive controls (PMA-treated Jurkat cells); use consistent cell densities; validate new antibody lots against previous ones

How can researchers distinguish between different phosphorylation patterns on SMAD2 in complex cellular contexts?

Distinguishing between different phosphorylation patterns on SMAD2 in complex cellular contexts requires sophisticated experimental approaches:

• Phospho-specific Antibody Panels:

  • Use antibodies targeting distinct phosphorylation sites (C-terminal sites vs. linker region sites like S255)

  • Apply these in parallel assays to create a phosphorylation profile

  • Quantify relative phosphorylation levels at different sites using densitometry or ELISA

• Phosphorylation Site Mutants:

  • Generate SMAD2 constructs with mutations at specific phosphorylation sites

  • Create single-site mutants and combinatorial mutants to assess interdependence

  • Express these in SMAD2-deficient backgrounds (CRISPR knockout cells)

  • Compare functional outcomes and phosphorylation at remaining sites

• Kinase Inhibitor Strategies:

  • Apply selective inhibitors targeting different kinases (TGF-β receptor kinases, NLK, CDKs, MAPKs)

  • Monitor changes in phosphorylation patterns to identify kinase-specific sites

  • Use time-course experiments to track sequential phosphorylation events

• Mass Spectrometry-Based Quantification:

  • Implement stable isotope labeling (SILAC) or TMT labeling for quantitative phosphoproteomics

  • Compare phosphopeptide abundance across different conditions

  • Analyze phosphorylation stoichiometry at multiple sites simultaneously

• Subcellular Fractionation:

  • Separate nuclear and cytoplasmic fractions to assess compartment-specific phosphorylation

  • Identify relationships between phosphorylation patterns and subcellular localization

  • Combine with immunofluorescence to visualize spatial distribution of differently phosphorylated SMAD2

By integrating these approaches, researchers can construct a comprehensive view of SMAD2 phosphorylation dynamics and their functional consequences in diverse signaling contexts .

What strategies can improve the reproducibility of experiments using Phospho-SMAD2 (S255) antibodies?

Ensuring reproducibility in experiments using Phospho-SMAD2 (S255) antibodies requires attention to multiple experimental parameters:

• Standardized Cell Culture Conditions:

  • Maintain consistent passage numbers (preferably p3-p10)

  • Control cell density at time of treatment (70-80% confluence recommended)

  • Standardize serum starvation protocols before stimulation

  • Document complete culture conditions (media composition, supplements, antibiotics)

• Rigorous Antibody Validation:

  • Perform titration experiments to determine optimal concentrations

  • Validate each new antibody lot against previous lots

  • Maintain detailed records of antibody source, catalog number, and lot

  • Include appropriate positive controls (PMA-treated Jurkat cells at 4 × 10^7 cells/ml)

• Controlled Stimulation Parameters:

  • Prepare fresh stimulants (TGF-β, PMA) for each experiment

  • Document precise stimulation duration and concentration

  • Use time-course experiments to identify optimal stimulation windows

  • Control temperature and CO2 conditions during treatments

• Standardized Sample Processing:

  • Develop and strictly follow detailed SOPs for sample collection and processing

  • Use consistent lysis buffer composition with freshly added protease/phosphatase inhibitors

  • Process all experimental conditions in parallel

  • Quantify protein concentration using reliable methods (BCA assay)

• Comprehensive Data Collection and Reporting:

  • Document all experimental parameters in laboratory notebooks

  • Include raw data and analysis methods in publications

  • Report both positive and negative results

  • Use technical and biological replicates (minimum n=3)

  • Apply appropriate statistical analyses

• Multi-technique Validation:

  • Confirm key findings using orthogonal techniques (IF, ELISA, Western blot)

  • Generate consistent results across different detection methods

  • Consider using phospho-ELISA kits that simultaneously measure total and phospho-SMAD2

How can Phospho-SMAD2 (S255) antibodies be utilized in cancer research?

Phospho-SMAD2 (S255) antibodies offer valuable tools for investigating TGF-β signaling dysregulation in cancer through multiple research applications:

• Biomarker Development:

  • Profile phospho-SMAD2 (S255) levels across different cancer types and stages

  • Correlate phosphorylation patterns with clinical outcomes and treatment responses

  • Develop prognostic or predictive indicators based on SMAD2 phosphorylation status

• Mechanistic Studies:

  • Investigate how altered kinase activities (NLK, CDKs) affect SMAD2 linker phosphorylation in cancer cells

  • Explore the relationship between SMAD2 phosphorylation and its tumor suppressor function in colorectal carcinoma

  • Examine how oncogenic pathways cross-talk with TGF-β signaling through SMAD2 phosphorylation

• Therapeutic Target Validation:

  • Assess how existing or experimental cancer therapeutics affect SMAD2 phosphorylation patterns

  • Screen compounds that selectively modulate linker region versus C-terminal phosphorylation

  • Evaluate combination treatments targeting both canonical and non-canonical SMAD2 phosphorylation pathways

• Resistance Mechanisms:

  • Characterize changes in SMAD2 phosphorylation associated with resistance to TGF-β pathway inhibitors

  • Identify compensatory phosphorylation events that maintain signaling despite therapeutic intervention

  • Develop strategies to overcome resistance based on comprehensive phosphorylation profiling

• Metastasis Research:

  • Track SMAD2 phosphorylation changes during epithelial-to-mesenchymal transition (EMT)

  • Correlate specific phosphorylation patterns with invasive and metastatic potential

  • Develop interventions targeting metastasis-promoting phosphorylation events

What insights can phosphorylation studies provide about the interplay between different kinases in regulating SMAD2 function?

Phosphorylation studies of SMAD2 reveal a complex regulatory network involving multiple kinases that coordinate to fine-tune TGF-β signaling:

• Kinase Network Interactions:

  • TGF-β receptor kinases primarily phosphorylate the C-terminal SXS motif, activating SMAD2

  • NLK phosphorylates the linker region (including S255), counteracting C-terminal phosphorylation

  • CDKs (cyclin-dependent kinases) can phosphorylate S245, S250, and S255 during cell cycle progression

  • MAPKs (ERK, JNK, p38) may target the linker region in response to various stimuli

  • GSK3β can phosphorylate SMAD2 following priming phosphorylation by other kinases

• Temporal Coordination:

  • Sequential phosphorylation events create a timing mechanism for signal duration

  • Initial C-terminal phosphorylation activates signaling

  • Subsequent linker phosphorylation prepares SMAD2 for degradation

  • This creates a self-limiting signal with defined duration

• Pathway Cross-talk:

  • Different kinases serve as integration points between TGF-β and other signaling pathways

  • NLK-mediated phosphorylation represents cross-talk with Wnt/β-catenin signaling

  • MAPK-mediated phosphorylation connects to growth factor and stress response pathways

  • CDK-mediated phosphorylation links TGF-β responses to cell cycle control

• Functional Outcomes:

  • Phosphorylation by different kinases can direct SMAD2 toward distinct transcriptional programs

  • Some phosphorylation patterns promote cytostatic responses, while others may enable pro-metastatic functions

  • The balance between various phosphorylation events determines the ultimate cellular response to TGF-β

Understanding this complex interplay provides insights into context-dependent TGF-β responses and opportunities for selective therapeutic targeting of specific branches of SMAD2 signaling .

How can genetic approaches complement antibody-based detection of phospho-SMAD2 in research?

Genetic approaches provide powerful complementary tools to antibody-based detection of phospho-SMAD2, enhancing research rigor and enabling unique experimental strategies:

• CRISPR/Cas9-mediated Gene Editing:

  • Generate SMAD2 knockout cell lines as negative controls for antibody validation

  • Create knock-in cell lines expressing SMAD2 with specific mutations at phosphorylation sites

  • Develop endogenously tagged SMAD2 (e.g., with fluorescent proteins) to track localization without antibodies

• Phosphorylation Site Mutants:

  • Express phospho-deficient (S→A) or phospho-mimetic (S→D/E) SMAD2 mutants

  • Create comprehensive mutation panels targeting individual or multiple phosphorylation sites

  • Rescue experiments in SMAD2-deficient backgrounds with various mutants

  • Example mutations:

    • S255A: prevents phosphorylation at the S255 site

    • S255D: mimics constitutive phosphorylation at S255

    • S245A/S250A/S255A: prevents phosphorylation across the linker region

• Reporter Systems:

  • Develop transcriptional reporters responsive to SMAD2 activity

  • Create split fluorescent protein systems that respond to SMAD2 phosphorylation state

  • Implement FRET-based biosensors to detect phosphorylation events in real-time

  • Correlate reporter output with antibody-based measurements

• Single-Cell Analysis:

  • Combine genetic reporters with single-cell sequencing to correlate SMAD2 activity with transcriptional outcomes

  • Perform lineage tracing to track long-term consequences of altered SMAD2 phosphorylation

  • Implement single-cell western techniques to correlate protein expression with phosphorylation status

• In Vivo Models:

  • Generate knock-in mouse models with phospho-site mutations in SMAD2

  • Create tissue-specific expression systems for SMAD2 variants

  • Develop conditional alleles to study temporal aspects of SMAD2 phosphorylation

These genetic approaches overcome several limitations of antibody-based detection, including specificity concerns, temporal resolution constraints, and the inability to establish causality between phosphorylation and function .