Product: Smad1/5/9 Antibody
Catalog: AF0614
Description: Rabbit polyclonal antibody to Smad1/5/9
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Zebrafish, Bovine, Sheep, Rabbit, Dog, Chicken, Xenopus
Mol.Wt.: 56kDa; 52kD(Calculated).
Uniprot: Q15797 | Q99717 | O15198
RRID: AB_2834266

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IF/ICC 1:100-1:500, IHC 1:50-1:200
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Prediction:
Pig(100%), Zebrafish(100%), Bovine(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(100%), Xenopus(100%)
Clonality:
Polyclonal
Specificity:
Smad1/5/9 Antibody detects endogenous levels of total Smad1/5/9.
RRID:
AB_2834266
Cite Format: Affinity Biosciences Cat# AF0614, RRID:AB_2834266.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

BSP-1; BSP1; HsMAD1; JV4-1; JV41; MAD homolog 1; MAD mothers against decapentaplegic homolog 1; Mad related protein 1; Mad-related protein 1; MADH1; MADR1; Mothers against decapentaplegic homolog 1; Mothers against DPP homolog 1; SMA- AND MAD-RELATED PROTEIN 1; SMAD 1; SMAD family member 1; SMAD mothers against DPP homolog 1; Smad1; SMAD1_HUMAN; TGF beta signaling protein 1; Transforming growth factor-beta-signaling protein 1; DKFZp781C1895; DKFZp781O1323; Dwfc; hSmad5; JV5 1; JV5-1; MAD homolog 5; MAD, mothers against decapentaplegic homolog 5; MADH 5; MADH5; Mothers against decapentaplegic homolog 5; mothers against decapentaplegic, drosophila, homolog of, 5; Mothers against DPP homolog 5; MusMLP; SMA and MAD related protein 5; SMAD 5; SMAD family member 5; SMAD, mothers against DPP homolog 5; Smad5; SMAD5_HUMAN; MAD homolog 9; Madh6; Mothers against decapentaplegic; Mothers against decapentaplegic homolog 9; Mothers against DPP homolog 9; SMAD 9; SMAD family member 9; SMAD, mothers against DPP homolog 9 (Drosophila); SMAD8A; SMAD8B; Smad9; SMAD9_HUMAN;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Expression:
Q15797 SMAD1_HUMAN:

Ubiquitous. Highest expression seen in the heart and skeletal muscle.

Q99717 SMAD5_HUMAN:

Ubiquitous.

O15198 SMAD9_HUMAN:

Expressed in heart, brain, placenta, lung, skeletal muscle, prostate, testis, ovary and small intestine. Also expressed in fetal brain, lung and kidney.

Description:
The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signals of the bone morphogenetic proteins (BMPs), which are involved in a range of biological activities including cell growth, apoptosis, morphogenesis, development and immune responses. In response to BMP ligands, this protein can be phosphorylated and activated by the BMP receptor kinase. The phosphorylated form of this protein forms a complex with SMAD4, which is important for its function in the transcription regulation. This protein is a target for SMAD-specific E3 ubiquitin ligases, such as SMURF1 and SMURF2, and undergoes ubiquitination and proteasome-mediated degradation. Alternatively spliced transcript variants encoding the same protein have been observed.
Sequence:
MNVTSLFSFTSPAVKRLLGWKQGDEEEKWAEKAVDALVKKLKKKKGAMEELEKALSCPGQPSNCVTIPRSLDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPLECCEFPFGSKQKEVCINPYHYKRVESPVLPPVLVPRHSEYNPQHSLLAQFRNLGQNEPHMPLNATFPDSFQQPNSHPFPHSPNSSYPNSPGSSSSTYPHSPTSSDPGSPFQMPADTPPPAYLPPEDPMTQDGSQPMDTNMMAPPLPSEINRGDVQAVAYEEPKHWCSIVYYELNNRVGEAFHASSTSVLVDGFTDPSNNKNRFCLGLLSNVNRNSTIENTRRHIGKGVHLYYVGGEVYAECLSDSSIFVQSRNCNYHHGFHPTTVCKIPSGCSLKIFNNQEFAQLLAQSVNHGFETVYELTKMCTIRMSFVKGWGAEYHRQDVTSTPCWIEIHLHGPLQWLDKVLTQMGSPHNPISSVS

MTSMASLFSFTSPAVKRLLGWKQGDEEEKWAEKAVDALVKKLKKKKGAMEELEKALSSPGQPSKCVTIPRSLDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPLDICEFPFGSKQKEVCINPYHYKRVESPVLPPVLVPRHNEFNPQHSLLVQFRNLSHNEPHMPQNATFPDSFHQPNNTPFPLSPNSPYPPSPASSTYPNSPASSGPGSPFQLPADTPPPAYMPPDDQMGQDNSQPMDTSNNMIPQIMPSISSRDVQPVAYEEPKHWCSIVYYELNNRVGEAFHASSTSVLVDGFTDPSNNKSRFCLGLLSNVNRNSTIENTRRHIGKGVHLYYVGGEVYAECLSDSSIFVQSRNCNFHHGFHPTTVCKIPSSCSLKIFNNQEFAQLLAQSVNHGFEAVYELTKMCTIRMSFVKGWGAEYHRQDVTSTPCWIEIHLHGPLQWLDKVLTQMGSPLNPISSVS

MHSTTPISSLFSFTSPAVKRLLGWKQGDEEEKWAEKAVDSLVKKLKKKKGAMDELERALSCPGQPSKCVTIPRSLDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPLECCEFPFGSKQKEVCINPYHYRRVETPVLPPVLVPRHSEYNPQLSLLAKFRSASLHSEPLMPHNATYPDSFQQPPCSALPPSPSHAFSQSPCTASYPHSPGSPSEPESPYQHSVDTPPLPYHATEASETQSGQPVDATADRHVVLSIPNGDFRPVCYEEPQHWCSVAYYELNNRVGETFQASSRSVLIDGFTDPSNNRNRFCLGLLSNVNRNSTIENTRRHIGKGVHLYYVGGEVYAECVSDSSIFVQSRNCNYQHGFHPATVCKIPSGCSLKVFNNQLFAQLLAQSVHHGFEVVYELTKMCTIRMSFVKGWGAEYHRQDVTSTPCWIEIHLHGPLQWLDKVLTQMGSPHNPISSVS

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Pig
100
Bovine
100
Sheep
100
Dog
100
Xenopus
100
Zebrafish
100
Chicken
100
Rabbit
100
Horse
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - Q15797/Q99717/O15198 As Substrate

Site PTM Type Enzyme
Ubiquitination
S11 Phosphorylation
K21 Ubiquitination
K32 Ubiquitination
S78 Phosphorylation
K81 Ubiquitination
Y88 Phosphorylation
K116 Sumoylation
K116 Ubiquitination
K118 Sumoylation
K118 Ubiquitination
S132 Phosphorylation
S144 Phosphorylation
Y146 Phosphorylation
S187 Phosphorylation P50750 (CDK9) , P49336 (CDK8) , P28482 (MAPK1)
S195 Phosphorylation P49336 (CDK8) , P50750 (CDK9) , P28482 (MAPK1)
T202 Phosphorylation P49841 (GSK3B)
S206 Phosphorylation P50750 (CDK9) , P28482 (MAPK1) , P49336 (CDK8) , P50613 (CDK7)
S210 Phosphorylation
S214 Phosphorylation P28482 (MAPK1) , P50750 (CDK9) , P49336 (CDK8)
S239 Phosphorylation Q13315 (ATM)
K269 Ubiquitination
S315 Phosphorylation
T322 Phosphorylation Q8N4C8 (MINK1) , Q9UKE5 (TNIK)
K418 Ubiquitination
S456 Phosphorylation
S462 Phosphorylation O00238 (BMPR1B)
S463 Phosphorylation Q05655 (PRKCD) , P36894 (BMPR1A) , O00238 (BMPR1B)
S465 Phosphorylation Q05655 (PRKCD) , O00238 (BMPR1B) , P36894 (BMPR1A)
Site PTM Type Enzyme
T2 Acetylation
S12 Phosphorylation
K22 Ubiquitination
K33 Ubiquitination
S57 Phosphorylation
S58 Phosphorylation
S63 Phosphorylation
K64 Ubiquitination
S79 Phosphorylation
K82 Ubiquitination
Y89 Phosphorylation
K117 Sumoylation
K119 Sumoylation
K119 Ubiquitination
Y128 Phosphorylation
S133 Phosphorylation
S152 Phosphorylation
S188 Phosphorylation
K306 Ubiquitination
S315 Phosphorylation
K418 Ubiquitination
S462 Phosphorylation
S463 Phosphorylation
S465 Phosphorylation
Site PTM Type Enzyme
K25 Ubiquitination
S40 Phosphorylation
S82 Phosphorylation
K85 Ubiquitination
Y92 Phosphorylation
T136 Phosphorylation Q16539 (MAPK14)
K159 Ubiquitination
S317 Phosphorylation
K420 Ubiquitination
S458 Phosphorylation
S464 Phosphorylation
S465 Phosphorylation
S467 Phosphorylation

Research Backgrounds

Function:

Transcriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase. SMAD1 is a receptor-regulated SMAD (R-SMAD). SMAD1/OAZ1/PSMB4 complex mediates the degradation of the CREBBP/EP300 repressor SNIP1. May act synergistically with SMAD4 and YY1 in bone morphogenetic protein (BMP)-mediated cardiac-specific gene expression.

PTMs:

Phosphorylation of the C-terminal SVS motif by BMP type 1 receptor kinase activates SMAD1 by promoting dissociation from the receptor and trimerization with SMAD4.

Ubiquitinated by SMAD-specific E3 ubiquitin ligase SMURF1, leading to its degradation. Monoubiquitinated, leading to prevent DNA-binding. Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes. Dephosphorylation, probably by PPM1A, induces its export from the nucleus to the cytoplasm (By similarity).

Subcellular Location:

Cytoplasm. Nucleus.
Note: Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4 (PubMed:15647271). Co-localizes with LEMD3 at the nucleus inner membrane (PubMed:15647271). Exported from the nucleus to the cytoplasm when dephosphorylated (By similarity).

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Ubiquitous. Highest expression seen in the heart and skeletal muscle.

Subunit Structure:

Found in a complex with SMAD4 and YY1. Interacts with HGS, NANOG and ZCCHC12 (By similarity). Upon C-terminus phosphorylation: forms trimers with another SMAD1 and the co-SMAD SMAD4. Interacts with PEBP2-alpha subunit, CREB-binding protein (CBP), p300, SMURF1, SMURF2, USP15 and HOXC8. Associates with ZNF423 or ZNF521 in response to BMP2 leading to activate transcription of BMP target genes. Interacts with SKOR1. Interacts (via MH2 domain) with LEMD3. Binding to LEMD3 results in at least a partial reduction of receptor-mediated phosphorylation. Forms a ternary complex with PSMB4 and OAZ1 before PSMB4 is incorporated into the 20S proteasome. Found in a macromolecular complex with FAM83G. Interacts (via MH2 domain) with FAM83G (via MH2 domain); in a SMAD4-independent manner. Interacts with ZC3H3 (By similarity). Interacts with TMEM119 (By similarity). Interacts (via MH1 and MH2 domains) with ZNF8 (By similarity). Interacts with RANBP3L; the interaction increases when SMAD1 is not phosphorylated and mediates SMAD1 nuclear export.

Family&Domains:

The MH2 domain mediates phosphorylation-dependent trimerization through L3 loop binding of phosphoserines in the adjacent subunit.

Belongs to the dwarfin/SMAD family.

Function:

Transcriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase. SMAD5 is a receptor-regulated SMAD (R-SMAD).

PTMs:

Phosphorylated on serine by BMP (bone morphogenetic proteins) type 1 receptor kinase.

Ubiquitin-mediated proteolysis by SMAD-specific E3 ubiquitin ligase SMURF1.

Subcellular Location:

Cytoplasm. Nucleus.
Note: Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Ubiquitous.

Subunit Structure:

May form trimers with the co-SMAD SMAD4. Interacts with PEBP2-alpha subunit and SMURF1. Interacts with SUV39H1 and SUV39H2. Interacts (via MH2 domain) with LEMD3. Interacts with WWP1. Interacts with TMEM119 (By similarity). Interacts with ZNF8. Interacts with RANBP3L.

Family&Domains:

Belongs to the dwarfin/SMAD family.

Function:

Transcriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase. SMAD9 is a receptor-regulated SMAD (R-SMAD).

PTMs:

Phosphorylated on serine by BMP (bone morphogenetic proteins) type 1 receptor kinase.

Subcellular Location:

Cytoplasm. Nucleus.
Note: In the cytoplasm in the absence of ligand. Migration to the nucleus when complexed with SMAD4 (By similarity).

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Expressed in heart, brain, placenta, lung, skeletal muscle, prostate, testis, ovary and small intestine. Also expressed in fetal brain, lung and kidney.

Subunit Structure:

Interaction with the co-SMAD SMAD4. Interacts with PEBP2-alpha subunit. Interacts with RANBP3L.

Family&Domains:

Belongs to the dwarfin/SMAD family.

Research Fields

· Cellular Processes > Cellular community - eukaryotes > Signaling pathways regulating pluripotency of stem cells.   (View pathway)

· Environmental Information Processing > Signal transduction > TGF-beta signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Hippo signaling pathway.   (View pathway)

· Human Diseases > Cancers: Overview > Transcriptional misregulation in cancer.

References

1). Zhu Y et al. Catalpol promotes the osteogenic differentiation of bone marrow mesenchymal stem cells via the Wnt/β-catenin pathway. Stem Cell Research & Therapy 2019 Jan 22;10(1):37 (PubMed: 30670092) [IF=7.5]

Application: WB    Species: rat    Sample: BMSCs

Additional file 2|:The effect of catalpol on chondrogenesis, adipogenesis, and the MAPK and BMP signalling of BMSCs was evaluated by WB. The data were confirmed by three repeated tests. The data are presented as the means ± SD. *P < 0.05 compared with the control group, #P < 0.05 compared with the 10 μM catalpol treatment group, ΔP < 0.05 compared with the 50 μM catalpol treatment group. (TIF 1433 kb)

2). Huang et al. TAZ promotes osteogenic differentiation of mesenchymal stem cells line C3H10T1/2, murine multi-lineage cells lines C2C12, and MEFs induced by BMP9. Cell Death Discovery [IF=7.0]

Application: WB    Species: Mouse    Sample: MSCs and MMCs

Fig. 5: The effect of TAZ on BMP9-induced classical Smad1/5/8 and Wnt/β-catenin in MSCs and MMCs.Cells were treated with TAZ or si-TAZ, followed by treatment with BMP9. A–L The total amount and phosphorylated forms of Smad1/5/8 were measured by western blot. A–L The total amount of β-catenin was measured by western blot. The data were shown as mean ± SD for three separated experiments. (*P 

3). Yan H et al. Kartogenin Improves Osteogenesis of Bone Marrow Mesenchymal Stem Cells via Autophagy. Stem Cells International 2022;2022(1278921) (PubMed: 36591373) [IF=4.3]

Application: WB    Species: Human    Sample: BMMSCs

Figure 4 KGN elevates autophagic activities in BMMSCs and prompts osteogenesis through Smad1/5/9 signal pathway. Western blotting (a) and semiquantification (b) of Smad and phosphorylation level of Smad1/5/9. 3-MA-mediated autophagy suppression inhibited KGN-induced Smad1/5/9 phosphorylation increase. ∗∗∗p < 0.001, one-way ANOVA test. Western blotting (c) and semiquantification (d) of the phosphorylation level of Smad1/5/9 in the control and LDN-treated BMMSCs. LDN effectively inhibited phosphorylation level of Smad 1/5/9. ∗∗p < 0.01, independent Student's t test. ARS detection (e) and semiquantification (f) of mineralized nodules. LDN inhibited KGN-induced osteogenesis. Scale bar = 500 μm. ∗p < 0.05 and ∗∗∗p < 0.001, one-way ANOVA test. (g) Western blotting, (h, i) semiquantification of (g), and (j, k) qRT-PCR assay of ALP and Runx2. LDN inhibited KGN-induced expression of osteogenic genes. ∗∗p < 0.01 and ∗∗∗p < 0.001, one-way ANOVA test. (l, m) Detection of ALP (green), Runx2 (green), and DAPI (blue) by immunofluorescent staining in BMMSCs. (n, o) Semiquantification of (l) and (m). LDN inhibited KGN-induced expression of osteogenic genes. Scale bar = 50 μm. ∗∗∗p < 0.001, one-way ANOVA test.

4). Yang L et al. Role of bone morphogenetic protein-2/4 in astrocyte activation in neuropathic pain. Molecular Pain 2019 Jan-Dec;15:1744806919892100 (PubMed: 31726923) [IF=3.3]

5). Liang et al. Disruption of the mouse Bmal1 locus promotes heterotopic ossification with aging via TGF-beta/BMP signaling. Journal of Bone and Mineral Metabolism 2022 Jan;40(1):40-55. (PubMed: 34626248) [IF=3.3]

6). Wang L et al. BMP6 regulates AMH expression via SMAD1/5/8 in goat ovarian granulosa cells. Theriogenology 2022 Nov 30;197(167-176) (PubMed: 36525856) [IF=2.8]

7). Zeng Y et al. Wnt and Smad signaling pathways synergistically regulated the osteogenic differentiation of fibroblasts in ankylosing spondylitis. TISSUE & CELL 2022 Jun 6;77:101852. (PubMed: 35753224) [IF=2.6]

8). Yang L et al. BMP2 increases hyperplasia and hypertrophy of bovine subcutaneous preadipocytes via BMP/SMAD signaling. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL 2022 Mar 11. (PubMed: 35275330) [IF=2.1]

9). Huang Y et al. Conductive nanostructured Si biomaterials enhance osteogeneration through electrical stimulation. Biomaterials Advances 2019 Oct;103:109748 (PubMed: 31349398)

Application: WB    Species: rat    Sample: rBMSCs

Fig. 9. |Electrical stimulation effect on the BMP2, BMP4 and Smad1/5/9 activation of rBMSCs cultured on micropyramid patterned Si surfaces with and without nanopores. (a) Images of RT-PCR product bands. (b)–(f) Analysis of image (a) using an image acquisition and analysis system. GAPDH was used to normalize the relative gene expression.

10). Yu Q et al. RON Mediates Tumor-Promoting Effects in Endometrial Adenocarcinoma. BioMed Research International 2021;2021:2282916 (PubMed: 34712728)

Application: WB    Species: Mouse    Sample: shRON cells

Figure 4 RON might play its role in endometrial adenocarcinoma cells through the Smad pathway. (a) Expression of p-smad1/5/9 in RON-OE cells. (b) Expression of p-smad1/5/9 in shRON cells. Values represent the mean ± SD, ∗P < 0.05. The assays were repeated three times.

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