Phospho-SMAD1 (S187) Antibody
Description
Target Overview
Phospho-SMAD1 (Ser187) is a transcription factor activated by bone morphogenetic protein (BMP) receptors. Phosphorylation at S187 modulates SMAD1’s nuclear translocation and interaction with co-regulators like SMAD4 to control gene expression during embryogenesis, cell differentiation, and tissue homeostasis . This modification is mediated by kinases such as CDK8/9 and MAPK1 .
Antibody Applications and Performance
Phospho-SMAD1 (S187) antibodies are validated for multiple applications across species:
Validation Data Highlights
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Western Blot:
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IHC:
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Specificity:
Cross-Reactivity Predictions
While validated for human, mouse, and rat, in silico analyses suggest potential reactivity with:
Research Implications
Phospho-SMAD1 (S187) antibodies enable studies on:
Product Specs
Target Background
- miR-26a-5p is highly expressed in synovial tissue of patients with RA, and its high expression can improve the invasive ability of synovial fibroblasts by targeting Smad 1 gene and accelerating the progression of RA. PMID: 30046030
- miR-23a facilitated cell proliferation and migration by targeting BMPR2/Smad1 signaling in hypoxia-induced human pulmonary artery smooth muscle cells. PMID: 29864909
- The expression of BMP15 in follicular fluid and Smad1 in granulosa cells was significantly decreased in the PCOS group compared with the control (P<0.05). This data suggested that the BMP15/Smad1 signaling pathway may be involved in granulosa cell apoptosis PMID: 28983616
- Mechanical stress affects the osteogenic differentiation of human ligamentum flavum cells via the BMP-Smad1 signaling pathway. PMID: 28944874
- Urinary Smad1 was associated with the degree of mesangial expansion in early diabetic nephropathy. PMID: 29490904
- Differential expression of TGF-beta superfamily members and role of Smad1/5/9-signaling in chondral versus endochondral chondrocyte differentiation. PMID: 27848974
- Uev1A appears to be involved in the BMP signaling pathway, where it collaborates with a ubiquitin E3 ligase Smurf1 to promote Smad1 degradation in a Ubc13-independent manner. PMID: 28771228
- Data show that miR-26b-5p suppresses Twist1-induced EMT, invasion, and metastasis of HCC cells by targeting SMAD1. PMID: 27027434
- Testosterone promoted tube formation of human umbilical endothelial cells, which was blocked by c-Src and ERK1/2 inhibitors or by the knockdown of Smad1. PMID: 28167128
- Low doses of IL1B activate the BMP/Smad signaling pathway to promote the osteogenesis of periodontal ligament stem cells, but higher doses of IL1B inhibit BMP/Smad signaling through the activation of NF-kappaB and MAPK signaling, inhibiting osteogenesis. PMID: 27415426
- Store operated calcium entry negatively regulates the Smad1 signaling pathway and inhibits Col IV protein production in glomerular mesangial cells. PMID: 28298362
- A significant association was found between the low expression of inhibitory protein SMAD-7 and both zeta-chain-associated protein kinase 70-negative cells (p = 0.04) and lower apoptotic index (p = 0.004). No differences were observed in SMAD-2/3 expression. In conclusion, our results demonstrate a significant correlation between greater SMAD-1/8 and lower SMAD-4 expression in chronic lymphocytic leukemia cells PMID: 28349818
- Melatonin treatment was found to downregulate TNFalpha-induced SMURF1 expression, subsequently decreasing SMURF1-mediated ubiquitination and degradation of SMAD1 protein PMID: 27265199
- The expression of specific targets Smad1 and Osterix was significantly increased in the presence of Pi and restored by coincubation with Mg(2+). As miR-30b, miR-133a, and miR-143 are negatively regulated by Pi and restored by Mg(2+) with a congruent modulation of their known targets Runx2, Smad1, and Osterix, our results provide a potential mechanistic explanation of the observed upregulation of these master switches of o PMID: 27419135
- The BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synth PMID: 27025722
- Regulation of impaired angiogenesis in diabetic dermal wound healing by microRNA-26a is mediated by the increased expression of its target gene, SMAD1. PMID: 26776318
- The expression SMAD1 protein showed a significant correlation with lung cancer differentiation and lymphatic metastasis (P < 0.05), but not with genders, ages, tumor sizes and histological types of lung cancer patients (P>0.05). PMID: 27049088
- Overexpression of Smad1 is associated with prostate cancer. PMID: 26227059
- SMAD1 signaling may be a key pathway contributing to the pathogenesis of Cardio-facio-cutaneous syndrome during early development. PMID: 25639853
- Smad1 elevation serves as a compensatory mechanism for p53 deficiency by potentiating the activation of p53 parallel pathways. PMID: 25757624
- Our data indicated that downregulation of miR-26b in osteosarcoma elevated the levels of CTGF and Smad1, facilitating osteosarcoma metastasis PMID: 25761878
- Smad1 acts as a novel binding protein of KSHV latency-associated nuclear antigen (LANA). LANA interacted with and sustained BMP-activated p-Smad1 in the nucleus and enhanced its loading on the Id promoters. PMID: 25010525
- Adult human Sertoli cells assumed similar morphological features, stable global gene expression profiles and numerous proteins, and activation of AKT and SMAD1/5 during long-period culture. PMID: 25880873
- The balance between Smad1/5- and Smad2/3-dependent signaling defines the outcome of the effect of TGF-beta on atherosclerosis where Smad1/5 is responsible for proatherogenic effects PMID: 25505291
- Data show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. PMID: 24850914
- Urinary Smad1 may be a potential diagnostic parameter for diabetic nephropathy and may be used to evaluate the severity of diabetic nephropathy PMID: 23943254
- Inhibiting Smurf1 mediated ubiquitination of Smad1/5. PMID: 24828823
- Smad1 is directly downregulated by miR-205. mRNA levels are not affected but Smad1 protein is decreased by miR-205 overexpression and increased by miR-205 inhibition. PMID: 23800974
- Results indicate that the BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride. PMID: 23918166
- Glucocorticoids recruit Tgfbr3 and Smad1 to shift transforming growth factor-beta signaling from the Tgfbr1/Smad2/3 axis to the Acvrl1/Smad1 axis in lung fibroblasts. PMID: 24347165
- The shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase. PMID: 24021264
- A detailed computational model for TGF-beta signaling that incorporates elements of previous models together with crosstalking between Smad1/5/8 and Smad2/3 channels through a negative feedback loop dependent on Smad7. PMID: 23804438
- Data indicate a transcription complex androgen receptor (AR)-p44-Smad1, and confirmed for physical interaction by co-immunoprecipitaion. PMID: 23734213
- Our studies establish that loss of SMAD1/5 leads to upregulation of PDGFA in ovarian granulosa cells PMID: 22964636
- Oscillatory shear stress induces synergistic interactions between specific BMPRs and integrin to activate Smad1/5 through the Shc/FAK/ERK pathway PMID: 23387849
- Shows the role of ALK-1 in many processes related to cardiovascular homeostasis, and the involvement of this protein in the development of cardiovascular diseases, suggesting the possibility of using the ALK-1/smad-1 pathway as a powerful therapeutic target PMID: 23707512
- TNF activated NF-kappaB pathway and inhibited the phosphorylation of Smad 1/5/8 and BMP-2-induced osteoblastic differentiation in BMMSCs PMID: 22897816
- Immunohistochemical analysis further revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. PMID: 23088614
- Immunohistochemical analysis of phosphorylated Smad1 showed nuclear expression in 70% of the osteosarcoma samples at levels comparable to osteoblastoma. Cases with lower expression showed significantly worse disease-free survival. PMID: 22868198
- Cav-1 is required and sufficient for Smad1 signaling in human dermal fibroblasts. PMID: 22277251
- Data suggest that Smads 1, 5 and 8 are potential prognostic markers and therapeutic targets for mTOR inhibition therapy of prostate cancer. PMID: 22452883
- TGF-beta induces the formation of complexes comprising phosphorylated Smad1/5 and Smad3, which bind to BMP-responsive elements in vitro and in vivo and mediate TGF-beta-induced transcriptional repression. PMID: 22615489
- Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow. PMID: 22550179
- Smurf1 is a negative feedback regulator for IFN-gamma signaling by targeting STAT1 for ubiquitination and proteasomal degradation. PMID: 22474288
- PAK2 negatively modulates TGF-beta signaling by attenuating the receptor-Smad interaction and thus Smad activation PMID: 22393057
- BMP-9 induced endothelial cell tubule formation and inhibition of migration involves Smad1 driven endothelin-1 production. PMID: 22299030
- Urinary Smad1 is a sensitive biomarker for diagnosis of diabetic glomerulosclerosis. PMID: 22073863
- Results show that BMP4-induced changes in OvCa cell morphology and motility are Smad-dependent with shRNA targeting Smads 1, 4, and 5. PMID: 21945631
- Expression of mutated Smad1 in adult human MSC cultures also resulted in increased nuclear accumulation of BMP-activated Smads and elevated gene transcripts characteristic of differentiating osteoblasts PMID: 21405981
- Endoglin promotes fibrosis in scleroderma fibroblasts via TGF-beta/Smad1 signaling. PMID: 21344387
Q&A
How does linker region phosphorylation differ from C-terminal phosphorylation of SMAD1?
SMAD1 undergoes two distinct phosphorylation events that have substantially different functional consequences:
| Phosphorylation Site | Kinase Responsible | Functional Outcome |
|---|---|---|
| C-terminal (S463/465) | BMP Type I receptors | Activation of SMAD1, complex formation with SMAD4, nuclear translocation, transcriptional activation |
| Linker region (S187) | MAPK/Erk pathway | Traditionally viewed as inhibitory, leading to degradation and cytoplasmic retention, but can also participate in pathway cross-talk and complex formation with other proteins like Xbra |
These differential phosphorylation events allow for integration of multiple signaling inputs. While C-terminal phosphorylation primarily activates canonical BMP signaling, linker region phosphorylation at sites including S187 enables context-dependent modulation of SMAD1 activity, creating a more nuanced cellular response .
What are the recommended dilutions and applications for Phospho-SMAD1 (S187) Antibody?
The Phospho-SMAD1 (S187) Antibody has been validated for multiple applications with specific recommended dilutions:
| Application | Recommended Dilution | Notes |
|---|---|---|
| Western Blot (WB) | 1:500-1:2000 | Higher sensitivity at 1:500 for detecting endogenous levels |
| Immunohistochemistry (IHC) | 1:50-1:300 | Optimal dilution varies by tissue type |
| ELISA | 1:10000 | Higher dilution appropriate for this sensitive method |
| Immunoprecipitation (IP) | 1:50-1:200 | Effective for protein complex studies |
| Immunocytochemistry (ICC) | 1:50-1:200 | For cellular localization studies |
When optimizing the antibody for your specific experimental system, it is advisable to test a range of dilutions starting with the manufacturer's recommendations .
What species reactivity has been confirmed for this antibody?
The Phospho-SMAD1 (S187) Antibody shows confirmed reactivity with multiple species:
| Species | Reactivity Status | Validation Method |
|---|---|---|
| Human | Confirmed | Western blot, IHC, multiple cell lines including HeLa |
| Mouse | Confirmed | Western blot (Raw264.7 cells) |
| Rat | Confirmed | Western blot (H9C2 cells) |
| Monkey | Confirmed | Cross-reactivity testing |
| Xenopus | Predicted | Based on sequence homology |
The antibody has been specifically tested in UV-treated cell lines across these species, making it versatile for comparative studies across different model systems .
How can I effectively preserve SMAD1 phosphorylation during sample preparation?
Preserving phosphorylation status is critical when working with phospho-specific antibodies. For Phospho-SMAD1 (S187), consider these methodological approaches:
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Buffer composition: Use lysis buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) at appropriate concentrations.
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Temperature control: Maintain samples at 4°C throughout processing to minimize phosphatase activity.
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Rapid processing: Minimize the time between tissue/cell collection and protein denaturation.
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Sample storage: For preservation of phosphorylated epitopes, store samples at -80°C with 50% glycerol in buffer containing phosphatase inhibitors.
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Stimulation controls: For positive control samples, treat cells with UV (4h) or BMP2, which have been shown to increase phosphorylation at S187 .
The detection of phosphorylated SMAD1 is highly sensitive to sample preparation conditions, and improper handling can lead to false negative results due to rapid dephosphorylation by endogenous phosphatases .
What are the best approaches for studying the interaction between MAPK/Erk and BMP signaling pathways using this antibody?
To effectively investigate the cross-talk between MAPK/Erk and BMP signaling using Phospho-SMAD1 (S187) Antibody:
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Pathway manipulation: Use pathway-specific activators (BMP2/4 for BMP pathway, FGF for MAPK/Erk pathway) and inhibitors (U0126 for MAPK/Erk pathway) to dissect specific contributions .
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Temporal analysis: Perform time-course experiments to track the dynamics of SMAD1 phosphorylation, as the timing of phosphorylation events at different sites is crucial for pathway integration.
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Co-immunoprecipitation: Combine the Phospho-SMAD1 (S187) Antibody with antibodies against pathway components (like SMAD4 or Xbra/Brachyury) to identify context-dependent protein complexes .
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Subcellular fractionation: Separate nuclear and cytoplasmic fractions to determine how phosphorylation affects SMAD1 localization.
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Mutational analysis: Compare wild-type SMAD1 with phospho-mimetic (S→D) or phospho-resistant (S→A) mutants in functional assays to determine the specific contribution of S187 phosphorylation .
Recent research has identified complex regulatory mechanisms involving Xbra (a transcription factor induced by FGF/Erk signaling), which can physically interact with linker-phosphorylated SMAD1 to form Xbra/SMAD1/SMAD4 trimeric complexes, leading to sustained nuclear localization of SMAD1 and protection from degradation .
How can Phospho-SMAD1 (S187) Antibody be used to study embryonic development and tissue specification?
The Phospho-SMAD1 (S187) Antibody is a valuable tool for studying developmental processes where BMP and MAPK pathway integration is critical:
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Mesoderm specification: This antibody can track how SMAD1 linker phosphorylation influences lateral mesoderm formation. Recent research in Xenopus embryos has shown that Xbra-dependent cooperative interplay between FGF/Erk and BMP/SMAD1 signaling is essential for lateral mesoderm specification .
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Tissue-specific signaling integration: Different embryonic tissues require specific levels of BMP activity, which can be monitored using the phospho-specific antibody. For example, somite formation may require moderate BMP/SMAD1 activity modulated by MAPK input, while ventral blood island formation may require higher BMP/SMAD1 activity .
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Temporal regulation: During critical developmental windows, the timing of phosphorylation events can determine cell fate decisions. The antibody allows researchers to track these events with temporal precision.
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In vivo models: The antibody has been successfully used in genetic models including mice with mutations that prevent phosphorylation of either the C-terminal motif or the MAPK motifs in the linker region, providing insights into the physiological significance of these phosphorylation events .
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Whole-mount immunostaining: For developmental studies in model organisms like Xenopus, whole-mount immunostaining with this antibody can reveal spatial patterns of pathway activation.
Of particular interest, recent research has shown that mice with mutations in SMAD1 linker phosphorylation sites exhibit defects in gastric epithelial homeostasis, primordial germ cell formation, and head and branchial arch development, underscoring the importance of MAPK-dependent SMAD1 phosphorylation in development .
What insights can be gained about SMAD1 stability and degradation using this antibody?
The Phospho-SMAD1 (S187) Antibody is particularly useful for investigating the complex regulation of SMAD1 stability and turnover:
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Ubiquitination studies: FGF/MAPK-mediated phosphorylation at S187 and other linker sites can prime SMAD1 for subsequent GSK3β-mediated phosphorylation, leading to polyubiquitination and proteasomal degradation. This antibody allows researchers to track this initial phosphorylation event .
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Protein half-life analysis: By combining cycloheximide (CHX) treatment with western blotting using this antibody, researchers can measure the half-life of phosphorylated SMAD1 under various conditions.
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Protective mechanisms: Recent research has identified that proteins like Xbra can protect linker-phosphorylated SMAD1 from degradation. This antibody has been used to demonstrate that Xbra maintains the levels of phospho-mimetic SMAD1 even in the presence of GSK3β .
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Subcellular localization: Linker phosphorylation can affect nuclear localization of SMAD1. Immunofluorescence with this antibody can reveal how S187 phosphorylation influences protein trafficking.
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Context-dependent regulation: The signaling outcome of S187 phosphorylation varies by cellular context, and this antibody helps reveal these differential effects across tissues and developmental stages.
In Xenopus embryos, researchers found that Xbra physically interacts with linker-phosphorylated SMAD1 to form a trimeric complex with SMAD4, which protects SMAD1 from ubiquitin-mediated proteasomal degradation and promotes its nuclear localization .
Why might I observe no signal or weak signal in Western blots with this antibody?
Several technical factors can lead to weak or absent signal when using Phospho-SMAD1 (S187) Antibody:
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Insufficient pathway activation: SMAD1 phosphorylation at S187 requires active MAPK/Erk signaling. Consider using positive controls with UV treatment (4 hours) or FGF stimulation, which have been demonstrated to increase this phosphorylation .
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Rapid dephosphorylation: Phosphorylation at S187 can be rapidly lost during sample preparation. Ensure your lysis buffer contains sufficient phosphatase inhibitors.
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Protein degradation: Linker-phosphorylated SMAD1 is targeted for degradation. Consider using proteasome inhibitors (MG132) in your experimental design.
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Inappropriate blocking agents: Some blocking agents can interfere with phospho-specific antibody binding. Try different blocking solutions (BSA versus milk).
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Antibody dilution: At 1:2000, signal may be weak for endogenous levels. Start with a 1:500 dilution for greater sensitivity .
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Storage conditions: Antibody efficacy can decrease with repeated freeze-thaw cycles. Store at -20°C or -80°C in small aliquots with 50% glycerol .
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Validation method: Consider running a phosphatase-treated control alongside your samples to confirm signal specificity.
If troubleshooting fails to resolve issues, manufacturers typically recommend confirming the presence of total SMAD1 protein first using a non-phospho-specific antibody before concluding there is an issue with the phospho-specific detection.
How can I distinguish between specific and non-specific signals when using this antibody?
Ensuring signal specificity is critical for accurate interpretation of results with Phospho-SMAD1 (S187) Antibody:
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Blocking peptide competition: Use the synthetic phosphopeptide derived from human SMAD1 around the phosphorylation site of S187 to compete with antibody binding. Signal that disappears with peptide competition is likely specific .
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Phosphatase treatment control: Treat a duplicate sample with lambda phosphatase before immunoblotting to demonstrate phosphorylation-dependent recognition.
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Pathway inhibition control: Treat cells with U0126 (MAPK/Erk inhibitor) to prevent S187 phosphorylation. Reduction or loss of signal confirms specificity to the MAPK-mediated phosphorylation .
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Mutant controls: If available, use SMAD1 S187A (phospho-resistant) mutant samples as negative controls.
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Molecular weight verification: Phosphorylated SMAD1 has an observed molecular weight of approximately 60 kDa, which may differ slightly from the calculated weight of 52 kDa due to post-translational modifications .
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Cross-reactivity testing: While this antibody is specific for phospho-S187 in SMAD1, it's important to note that it does not cross-react with other proteins but may recognize the same phosphorylated motif in the highly homologous SMAD5 and SMAD8 proteins .
For immunohistochemistry applications, always include a negative control using the secondary antibody alone or PBS instead of the primary antibody to identify any non-specific background staining .
