Phospho-SMAD3 (Ser425) Antibody
Description
Molecular Basis of SMAD3 Phosphorylation
SMAD3 functions as a receptor-regulated SMAD (R-SMAD) that serves as an intracellular signal transducer and transcriptional modulator within the TGF-β signaling pathway. Following stimulation by TGF-β, SMAD3 becomes phosphorylated at carboxyl terminal serine residues (Ser423 and Ser425) by TGF-β Receptor I . This phosphorylation represents a critical activation step that enables SMAD3 to form complexes with SMAD4 and translocate to the nucleus, where it regulates the expression of target genes involved in diverse cellular processes .
The specificity of this phosphorylation event is particularly noteworthy. While SMAD3 contains multiple phosphorylation sites, including those in the linker region (Thr-179, Ser-204, and Ser-208) that can be phosphorylated by various kinases such as glycogen synthase kinase 3 (GSK3), the C-terminal phosphorylation at Ser423 and Ser425 is exclusively mediated by the TGF-β receptor I kinase . This distinctive modification serves as a molecular switch that shifts SMAD3 from an inactive to an active state, fundamentally altering its binding partnerships and subcellular localization.
Characteristics of Phospho-SMAD3 (Ser425) Antibodies
Phospho-SMAD3 (Ser425) antibodies are specifically designed to recognize SMAD3 only when phosphorylated at the Ser425 residue. These antibodies are available in various formats, including monoclonal and polyclonal variants, each offering distinct advantages for different experimental applications.
Specificity and Cross-Reactivity
Cross-species reactivity analyses based on sequence homology suggest that many Phospho-SMAD3 (Ser425) antibodies can detect the phosphorylated protein across multiple species, including human, mouse, rat, pig, dog, cow, horse, chicken, and even Xenopus species . This high degree of conservation in the phosphorylation site and surrounding sequence enables broad application of these antibodies across different model systems.
Applications and Protocols
Phospho-SMAD3 (Ser425) antibodies find utility in a wide range of experimental techniques aimed at investigating TGF-β signaling pathways and SMAD3 activation states.
Common Applications and Recommended Dilutions
| Application | Dilution Range | Protocol Notes |
|---|---|---|
| Western Blotting | 1:500 - 1:1000 | Detect bands at ~52-60 kDa |
| Immunoprecipitation | 1:50 | Requires 5 μl antibody per sample |
| Chromatin IP | 1:100 | Use with 10 μg chromatin (~4×10⁶ cells) |
| Immunohistochemistry | 1:50 - 1:100 | Works with paraffin and frozen sections |
| Immunofluorescence | 1:50 - 1:250 | Shows nuclear translocation upon TGF-β treatment |
| ELISA | Varies by kit | Quantitative detection of phosphorylation |
| Flow Cytometry | Varies by antibody | Typically requires conjugated antibodies |
For optimal results in Western blotting, the antibodies effectively detect endogenous levels of phosphorylated SMAD3 in stimulated cells, typically appearing as bands at approximately 52-60 kDa . In chromatin immunoprecipitation assays, these antibodies can identify genomic regions occupied by phosphorylated SMAD3, providing insights into transcriptional regulation mechanisms .
Cell-Based Phosphorylation Assays
Cell-based colorimetric ELISA kits utilizing Phospho-SMAD3 (Ser425) antibodies offer a convenient, lysate-free approach for monitoring SMAD3 phosphorylation in intact cells . These assays typically involve:
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Cell culture and treatment in microplates
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Fixation and permeabilization
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Incubation with primary Phospho-SMAD3 (Ser425) antibody
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Detection with enzyme-conjugated secondary antibody
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Colorimetric or fluorescent readout
Such assays provide a high-throughput method for screening compounds that modulate TGF-β signaling or for measuring pathway activation in various experimental conditions.
Product Comparison
| Manufacturer | Catalog Number | Clone/Type | Validated Applications | Special Features |
|---|---|---|---|---|
| Cell Signaling | #9520 | C25A9/Rabbit mAb | WB, IP, ChIP | Recombinant production for lot consistency |
| Affinity Biosciences | AF3362 | Rabbit polyclonal | WB, IHC, IF/ICC | Cross-reactivity with multiple species |
| Santa Cruz | sc-517575 | 1D9/Mouse mAb | IF, IHC-P | Cited in 32 publications |
| Bio-Techne | NBP1-77836 | Rabbit polyclonal | WB, IHC, ICC, ELISA | Available with fluorescent conjugates |
| Thermo Fisher | MA5-14936 | S.434.0/Monoclonal | WB, IHC, IF | High specificity for phospho-form |
Modern production methods, particularly for recombinant antibodies, offer superior lot-to-lot consistency, continuous supply, and animal-free manufacturing processes . These advances have significantly improved the reliability of experimental results obtained with Phospho-SMAD3 (Ser425) antibodies.
Advanced Detection Technologies
Beyond traditional antibody formats, innovative detection technologies have emerged to enhance the sensitivity and versatility of Phospho-SMAD3 (Ser425) analysis. The HTRF (Homogeneous Time-Resolved Fluorescence) Human & Mouse Phospho-SMAD3 (Ser423/425) Detection Kit represents one such advancement, offering a no-wash assay format that utilizes two labeled antibodies: one specific for the phosphorylated motif and another recognizing the protein regardless of phosphorylation status . When both antibodies bind to phosphorylated SMAD3, the close proximity of their fluorophores generates a FRET signal proportional to the concentration of phosphorylated protein.
Biological Significance and Research Applications
The phosphorylation of SMAD3 at Ser425 represents a critical event in TGF-β signal transduction with far-reaching implications for cellular physiology and pathology.
Role in TGF-β Signaling Pathway
Phosphorylation at Ser425 (along with Ser423) by TGF-β Receptor I kinase serves as the principal activation mechanism for SMAD3. This modification enables:
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Dissociation from cytoplasmic anchoring proteins
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Complex formation with SMAD4
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Nuclear translocation
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Binding to specific DNA elements (typically TRE elements)
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Recruitment of transcriptional co-factors like p300/CBP
Through these mechanisms, phosphorylated SMAD3 mediates many of the cellular responses to TGF-β, including cell cycle arrest, apoptosis, differentiation, extracellular matrix production, and immune regulation.
Regulatory Mechanisms and Feedback Control
Research has revealed sophisticated mechanisms that regulate SMAD3 phosphorylation at Ser425. A study published in Nature Communications demonstrated that TGF-β can induce phosphorylation at multiple sites in SMAD3, including the linker region (Thr-179, Ser-204, and Ser-208) in addition to the C-terminal residues . Interestingly, mutations at Ser-204 and Ser-208 were found to strengthen the transcriptional activity of SMAD3 by enhancing its affinity for the co-activator CBP, suggesting a negative feedback control mechanism .
More recent research has explored innovative approaches to modulate SMAD3 phosphorylation. One study demonstrated targeted dephosphorylation of phospho-SMAD3 using a proximity-induced phosphatase system, offering a potential modality for rewiring cell signaling . This approach significantly decreased TGF-β-induced transcription of SMAD3-responsive genes, including SERPINE-1 and SMAD7.
Future Directions and Emerging Applications
The development and application of Phospho-SMAD3 (Ser425) antibodies continue to evolve, opening new avenues for research and potential therapeutic interventions.
Advanced Imaging Applications
Fluorescently conjugated Phospho-SMAD3 (Ser425) antibodies, such as those labeled with mFluor Violet 450 SE (excitation 406 nm, emission 445 nm), enable sophisticated imaging applications for visualizing the dynamics of SMAD3 activation and nuclear translocation in live or fixed cells . These tools provide spatial and temporal resolution for studying TGF-β signaling events.
Therapeutic Implications
Understanding the regulation of SMAD3 phosphorylation has significant implications for developing therapeutic strategies targeting TGF-β signaling in diseases such as fibrosis, cancer, and autoimmune disorders. Phospho-SMAD3 (Ser425) antibodies serve as valuable research tools for evaluating the efficacy of potential inhibitors or modulators of this pathway .
Recent studies have explored approaches for targeted manipulation of SMAD3 phosphorylation status as a potential therapeutic strategy. The ability to selectively dephosphorylate SMAD3 at Ser425 offers promising avenues for intervening in pathological TGF-β signaling without disrupting other essential functions of this multifaceted pathway .
Product Specs
Target Background
- A study established a relationship between OCT4 and SMAD3 heterodimers formation and the promotion of Snail, Slug, and CXCL13 transcription, ultimately contributing to breast cancer progression. PMID: 29526821
- Gene editing studies indicate the cancer-promoting role of Smad3 T179 phosphorylation in human triple-negative breast cancer cells. PMID: 30251686
- Downregulation of miR-637 promotes proliferation and migration of fibroblasts by targeting Smad3 in keloids. PMID: 29845237
- Findings suggest that miR326 inhibits endometrial fibrosis by suppressing the TGFbeta1/Smad3 signaling pathway, indicating that miR326 may be a prognostic biomarker and therapeutic target for Intrauterine adhesion (IUA). PMID: 29956752
- A study validated a specific model prediction that SMAD3 regulates Huntington's disease (HD)-related gene expression changes. Additionally, results revealed CAG repeat length-dependent changes in the genomic occupancy of SMAD3 and confirmed the model's prediction that many SMAD3 target genes are downregulated early in HD. PMID: 29581148
- The SMAD3 rs12901499 polymorphism may be involved in the development of knee osteoarthritis. Larger studies with more diverse ethnic populations are needed to confirm these findings. PMID: 29315792
- NLRC5 may act as a key mediator in renal fibroblast activation and fibrogenesis. PMID: 29608899
- The SMAD3 SNP rs12901499 GA genotype and G variant may increase the risk of hip osteoarthritis in Chinese Han patients. PMID: 29310478
- Positive cooperativity of Smad3 and STAT3 during epithelial-mesenchymal transition [Review]. PMID: 29140406
- CXCL12 activates the MEKK1/JNK signaling pathway, which in turn initiates SMAD3 phosphorylation, its translocation to nuclei, and recruitment of SMAD3 to the CTGF promoter, ultimately inducing CTGF expression in human lung fibroblasts. PMID: 29499695
- These results indicated that Bone marrow-derived mesenchymal stem cells -conditioned medium suppressed the epithelial-mesenchymal transition, which might be associated with TGF-B1/Smad3. This study provides a theoretical foundation for research into the mechanisms responsible for pulmonary disease. PMID: 29207055
- The present findings indicate that RACK1 silencing attenuates renal fibrosis by suppressing the activation of the TGF-beta1/Smad3 signaling pathway in HK-2 cells. Therefore, RACK1 may serve as a novel regulator of renal fibrosis. PMID: 29039466
- MSP analysis from 81 Acute coronary syndrome (ACS) samples, 74 SCAD samples and 53 healthy samples, and Sequenom MassARRAY analysis, confirmed that differential CpG methylation of SMAD3 was significantly corrected with the reference results of the HumanMethylation450 array. PMID: 29115576
- Smad3 knockdown could restore the inhibition of cell proliferation induced by FSTL1 overexpression in MDAMB231FSTL1 cells, indicating that the antiproliferative effect of FSTL1 overexpression may be associated with Smad3-involved TGFbeta signaling pathway regulation. This study identified FSTL1 as an inhibitor of cell proliferation in MDAMB231 and 231BR cell lines. PMID: 29048681
- miR-195 inhibited proliferation and induced apoptosis of vascular smooth muscle cells, which was abated by Smad3 overexpression. PMID: 28665537
- SMAD3 SNP rs422342 is statistically associated with intervertebral disc degeneration in the Greek population. PMID: 28662992
- We observed that SMAD3 rs1065080 single nucleotide gene polymorphisms were significantly associated with patient susceptibility to intracranial arterial aneurysms. PMID: 28988651
- Smad3 binds with type I TGF-beta receptor (TRI) even in unstimulated cells. PMID: 27641076
- This study demonstrates that Smad3 protein had low expression in ACTH-Pituitary Adenoma Development. PMID: 29524699
- Data suggest that TGF-beta stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1, and the three MAPK signaling pathways. PMID: 28608941
- These results suggested that FXR may serve as an important negative regulator for manipulating Smad3 expression, and the FXR/Smad3 pathway may be a novel target for the treatment of renal fibrosis. PMID: 27853248
- SMad3 role in TGF-beta/SMAD pathway signal transduction. PMID: 28320972
- ERK1/2 mediates Heme oxygenase-1 or CO-induced Smad3 phosphorylation at Thr179. PMID: 29524413
- Participants' data and peripheral blood samples were collected, and three Smad3 CpG loci were examined. Smad3 mRNA expression was significantly higher in the patient group than in the negative control group but did not differ between the two control groups. PMID: 28562330
- The critical roles of the miR-16-5p-Smad3 pathway in melatonin-induced growth defects of gastric cancers. PMID: 29359963
- TGFbeta1 signaling is associated with the activation of SMAD3 at the ciliary base. PMID: 27748449
- Exaggerated WNT-5B expression upon cigarette smoke exposure in the bronchial epithelium of COPD patients leads to TGF-beta/Smad3-dependent expression of genes related to airway remodeling. PMID: 27126693
- HSF1 activity is decreased in fibrotic hearts. HSF1 inhibits phosphorylation and nuclear distribution of Smad3 via direct binding to Smad3. Active Smad3 blocks the anti-fibrotic effect of HSF1. PMID: 28091697
- miR-142-5p plays as a negative regulator in the TGF-beta pathway by targeting SMAD3 and suppresses TGF-beta-induced growth inhibition in cancer cells. PMID: 27683030
- Authors were able to confirm the expression of SMAD3 in intact and degraded cartilage of the knee and hip. Our findings provide the first systematic evaluation of pleiotropy between OA and BMD, highlight genes with biological relevance to both traits, and establish a robust new OA genetic risk locus at SMAD3. PMID: 28934396
- A bioinformatics analysis and luciferase reporter assay identified Smad3 as a direct target gene of miR-216b, and Smad3 expression was reduced by miR-216b overexpression at both the mRNA and protein levels. PMID: 28356485
- Because the expression of these genes correlates with cell shape, these are likely mechanosensitive genes that regulate SMAD3 and/or RELA activation in response to mechanical cues. PMID: 27864353
- SMAD3 transcription factor binds RNA with large internal loops or bulges with high apparent affinity, suggesting a biological role for RNA binding by SMAD3. PMID: 29036649
- Case Report: internal mammary artery aneurysms in sisters with SMAD3 mutation. PMID: 28286188
- High Smad3 expression is associated with invasion and metastasis in pancreatic ductal adenocarcinoma. PMID: 26908446
- New evidence suggests that SMAD3 activation may serve as a critical converging point of dysregulated TGFB superfamily signaling and genetic aberrations in human granulosa cell tumor development (review). PMID: 27683263
- We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. PMID: 27705785
- ANP inhibits TGF-beta1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-beta1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment of pulmonary diseases with airway remodeling. PMID: 28229930
- Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression through CBP, thereby mediating the epithelial-mesenchymal transition. PMID: 27769780
- Particularly, galangin effectively inhibits phosphorylation of the Thr-179 site at the Smad3 linker region through suppression of CDK4 phosphorylation. Thus, galangin can be a promising candidate as a selective inhibitor to suppress phosphorylation of the Smad3 linker region. PMID: 29097203
- Up-regulation of miR-195 suppressed cell migration and invasion in vitro. Smad3 was verified as a direct target of miR-195, which was further confirmed by the inverse expression of miR-195 and Smad3 in patients' specimens. PMID: 27206216
- In human primary tubular epithelial cells, inhibition of HIF sensing prolylhydroxylases by DMOG or exposure of the cells to hypoxia upregulated Smad3 expression and enhanced its translocation to the nucleus. PMID: 27155083
- Findings demonstrate that TGFbeta1 allows tumors to evade host immune responses in part through enhanced SMAD3-mediated PD-1 expression on tumor infiltrating lymphocytes. PMID: 27683557
- Store-operated calcium entry via Orai1 in mesangial cells negatively regulates the TGF-beta1/Smad3 signaling pathway. PMID: 28637791
- TF-induced microvessel stabilization is regulated via PAR2-SMAD3, which is indispensable for the maintenance of vascular integrity. PMID: 26658897
- Establish PPM1A as a novel repressor of the SMAD3 pathway in renal fibrosis. PMID: 27328942
- Methylation in SMAD3 was selectively increased in asthmatic children of asthmatic mothers and was associated with childhood asthma risk. PMID: 28011059
- A direct crosstalk between the STAT3 and Smad3 signaling pathways that may contribute to tumor development and inflammation. PMID: 26616859
- It is reported here that TGF-beta directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated threonine179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. PMID: 27746021
- Bcl-3 knockdown enhanced the degradation of Smad3 but not Smad2 following TGFbeta treatment. PMID: 27906182
