Product: Smad3 Antibody
Catalog: AF6362
Source: Rabbit
Application: WB, IHC, IF/ICC, ELISA(peptide)
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep, Rabbit, Dog, Chicken, Xenopus
Mol.Wt.: 58kD; 48kD(Calculated).
Uniprot: P84022
RRID: AB_2835210

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500, ELISA(peptide) 1:20000-1:40000
*The optimal dilutions should be determined by the end user.
Reactivity:
Human,Mouse,Rat
Prediction:
Pig(100%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(100%), Xenopus(100%)
Clonality:
Polyclonal
Specificity:
Smad3 Antibody detects endogenous levels of total Smad3.
RRID:
AB_2835210
Cite Format: Affinity Biosciences Cat# AF6362, RRID:AB_2835210.
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

DKFZP586N0721; DKFZp686J10186; hMAD 3; hMAD-3; hSMAD3; HSPC193; HST17436; JV15 2; JV15-2; JV152; LDS1C; LDS3; MAD (mothers against decapentaplegic Drosophila) homolog 3; MAD homolog 3; Mad homolog JV15 2; Mad protein homolog; MAD, mothers against decapentaplegic homolog 3; Mad3; MADH 3; MADH3; MGC60396; Mothers against decapentaplegic homolog 3; Mothers against DPP homolog 3; SMA and MAD related protein 3; SMAD 3; SMAD; SMAD family member 3; SMAD, mothers against DPP homolog 3; Smad3; SMAD3_HUMAN;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Description:
Smad3 transcription factor phosphorylated and activated by TGF-beta-type receptors. A receptor-regulated Smad (R-smad). Binds directly to consensus DNA-binding elements in the promoters of target genes. In mouse required for establishemnt of the mucosal immune response and proper development of skeleton.
Sequence:
MSSILPFTPPIVKRLLGWKKGEQNGQEEKWCEKAVKSLVKKLKKTGQLDELEKAITTQNVNTKCITIPRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELRAMELCEFAFNMKKDEVCVNPYHYQRVETPVLPPVLVPRHTEIPAEFPPLDDYSHSIPENTNFPAGIEPQSNIPETPPPGYLSEDGETSDHQMNHSMDAGSPNLSPNPMSPAHNNLDLQPVTYCEPAFWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS

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

PTMs - P84022 As Substrate

Site PTM Type Enzyme
Ubiquitination
S2 Acetylation
S3 Phosphorylation
T8 Phosphorylation P11802 (CDK4) , P24941 (CDK2)
K13 Ubiquitination
K19 Acetylation
K29 Acetylation
K33 Ubiquitination
K36 Sumoylation
S37 Phosphorylation
K63 Ubiquitination
T66 Phosphorylation P49841 (GSK3B)
S78 Phosphorylation
K81 Ubiquitination
Y88 Phosphorylation
Y125 Phosphorylation
T132 Phosphorylation
T179 Phosphorylation P49336 (CDK8) , P28482 (MAPK1) , P24941 (CDK2) , P50750 (CDK9) , P11802 (CDK4) , P31749 (AKT1)
S204 Phosphorylation P28482 (MAPK1) , Q14680 (MELK) , Q16539 (MAPK14) , P27361 (MAPK3) , P11802 (CDK4)
S208 Phosphorylation P49336 (CDK8) , P11802 (CDK4) , P28482 (MAPK1) , Q16539 (MAPK14) , P50750 (CDK9)
S213 Phosphorylation P50750 (CDK9) , P49336 (CDK8) , P11802 (CDK4) , P28482 (MAPK1) , P24941 (CDK2)
S275 Phosphorylation
S309 Phosphorylation
K378 Acetylation
T388 Phosphorylation
T412 Phosphorylation
S416 Phosphorylation
S418 Phosphorylation P78368 (CSNK1G2)
S422 Phosphorylation P36897 (TGFBR1)
S423 Phosphorylation P36897 (TGFBR1)
S425 Phosphorylation P36897 (TGFBR1)

Research Backgrounds

Function:

Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD3/SMAD4 complex, activates transcription. Also can form a SMAD3/SMAD4/JUN/FOS complex at the AP-1/SMAD site to regulate TGF-beta-mediated transcription. Has an inhibitory effect on wound healing probably by modulating both growth and migration of primary keratinocytes and by altering the TGF-mediated chemotaxis of monocytes. This effect on wound healing appears to be hormone-sensitive. Regulator of chondrogenesis and osteogenesis and inhibits early healing of bone fractures. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.

PTMs:

Phosphorylated on serine and threonine residues. Enhanced phosphorylation in the linker region on Thr-179, Ser-204 and Ser-208 on EGF and TGF-beta treatment. Ser-208 is the main site of MAPK-mediated phosphorylation. CDK-mediated phosphorylation occurs in a cell-cycle dependent manner and inhibits both the transcriptional activity and antiproliferative functions of SMAD3. This phosphorylation is inhibited by flavopiridol. Maximum phosphorylation at the G(1)/S junction. Also phosphorylated on serine residues in the C-terminal SXS motif by TGFBR1 and ACVR1. TGFBR1-mediated phosphorylation at these C-terminal sites is required for interaction with SMAD4, nuclear location and transactivational activity, and appears to be a prerequisite for the TGF-beta mediated phosphorylation in the linker region. Dephosphorylated in the C-terminal SXS motif by PPM1A. This dephosphorylation disrupts the interaction with SMAD4, promotes nuclear export and terminates TGF-beta-mediated signaling. Phosphorylation at Ser-418 by CSNK1G2/CK1 promotes ligand-dependent ubiquitination and subsequent proteasome degradation, thus inhibiting SMAD3-mediated TGF-beta responses. Phosphorylated by PDPK1.

Acetylation in the nucleus by EP300 in the MH2 domain regulates positively its transcriptional activity and is enhanced by TGF-beta.

Poly-ADP-ribosylated by PARP1 and PARP2. ADP-ribosylation negatively regulates SMAD3 transcriptional responses during the course of TGF-beta signaling.

Ubiquitinated. Monoubiquitinated, leading to prevent DNA-binding. Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes. Ubiquitinated by RNF111, leading to its degradation: only SMAD3 proteins that are 'in use' are targeted by RNF111, RNF111 playing a key role in activating SMAD3 and regulating its turnover (By similarity). Undergoes STUB1-mediated ubiquitination and degradation.

Subcellular Location:

Cytoplasm. Nucleus.
Note: Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4 (PubMed:15799969). Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1 (PubMed:16751101, PubMed:19289081). Co-localizes with LEMD3 at the nucleus inner membrane (PubMed:15601644). MAPK-mediated phosphorylation appears to have no effect on nuclear import (PubMed:19218245). PDPK1 prevents its nuclear translocation in response to TGF-beta (PubMed:17327236).

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

Monomer; in the absence of TGF-beta. Homooligomer; in the presence of TGF-beta. Heterotrimer; forms a heterotrimer in the presence of TGF-beta consisting of two molecules of C-terminally phosphorylated SMAD2 or SMAD3 and one of SMAD4 to form the transcriptionally active SMAD2/SMAD3-SMAD4 complex. Interacts with TGFBR1. Part of a complex consisting of AIP1, ACVR2A, ACVR1B and SMAD3. Interacts with AIP1, TGFB1I1, TTRAP, FOXL2, PML, PRDM16, HGS, WWP1 and SNW1. Interacts (via MH2 domain) with CITED2 (via C-terminus). Interacts with NEDD4L; the interaction requires TGF-beta stimulation. Interacts (via MH2 domain) with ZFYVE9. Interacts with HDAC1, TGIF and TGIF2, RUNX3, CREBBP, SKOR1, SKOR2, SNON, ATF2, SMURF2 and TGFB1I1. Interacts with DACH1; the interaction inhibits the TGF-beta signaling. Forms a complex with SMAD2 and TRIM33 upon addition of TGF-beta. Found in a complex with SMAD3, RAN and XPO4. Interacts in the complex directly with XPO4. Interacts (via MH2 domain) with LEMD3; the interaction represses SMAD3 transcriptional activity through preventing the formation of the heteromeric complex with SMAD4 and translocation to the nucleus. Interacts with RBPMS. Interacts (via MH2 domain) with MECOM. Interacts with WWTR1 (via its coiled-coil domain). Interacts (via the linker region) with EP300 (C-terminal); the interaction promotes SMAD3 acetylation and is enhanced by TGF-beta phosphorylation in the C-terminal of SMAD3. This interaction can be blocked by competitive binding of adenovirus oncoprotein E1A to the same C-terminal site on EP300, which then results in partially inhibited SMAD3/SMAD4 transcriptional activity. Interacts with SKI; the interaction represses SMAD3 transcriptional activity. Component of the multimeric complex SMAD3/SMAD4/JUN/FOS which forms at the AP1 promoter site; required for synergistic transcriptional activity in response to TGF-beta. Interacts (via an N-terminal domain) with JUN (via its basic DNA binding and leucine zipper domains); this interaction is essential for DNA binding and cooperative transcriptional activity in response to TGF-beta. Interacts with PPM1A; the interaction dephosphorylates SMAD3 in the C-terminal SXS motif leading to disruption of the SMAD2/3-SMAD4 complex, nuclear export and termination of TGF-beta signaling. Interacts (dephosphorylated form via the MH1 and MH2 domains) with RANBP3 (via its C-terminal R domain); the interaction results in the export of dephosphorylated SMAD3 out of the nucleus and termination of the TGF-beta signaling. Interacts with MEN1. Interacts with IL1F7. Interaction with CSNK1G2. Interacts with PDPK1 (via PH domain). Interacts with DAB2; the interactions are enhanced upon TGF-beta stimulation. Interacts with USP15. Interacts with PPP5C; the interaction decreases SMAD3 phosphorylation and protein levels. Interacts with LDLRAD4 (via the SMAD interaction motif). Interacts with PMEPA1. Interacts with ZC3H3 (By similarity). Interacts with ZNF451. Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4. Interacts with ZFHX3. Interacts weakly with ZNF8. Interacts (when phosphorylated) with RNF111; RNF111 acts as an enhancer of the transcriptional responses by mediating ubiquitination and degradation of SMAD3 inhibitors (By similarity). Interacts with STUB1, HSPA1A, HSPA1B, HSP90AA1 and HSP90AB1. Interacts (via MH2 domain) with ZMIZ1 (via SP-RING-type domain); in the TGF-beta signaling pathway increases the activity of the SMAD3/SMAD4 transcriptional complex.

Family&Domains:

The MH1 domain is required for DNA binding. Also binds zinc ions which are necessary for the DNA binding.

The MH2 domain is required for both homomeric and heteromeric interactions and for transcriptional regulation. Sufficient for nuclear import.

The linker region is required for the TGFbeta-mediated transcriptional activity and acts synergistically with the MH2 domain.

Belongs to the dwarfin/SMAD family.

Research Fields

· Cellular Processes > Cell growth and death > Cell cycle.   (View pathway)

· Cellular Processes > Transport and catabolism > Endocytosis.   (View pathway)

· Cellular Processes > Cell growth and death > Cellular senescence.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Adherens junction.   (View pathway)

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

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

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

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

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

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

· Human Diseases > Infectious diseases: Parasitic > Chagas disease (American trypanosomiasis).

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > HTLV-I infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Colorectal cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Pancreatic cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Chronic myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Hepatocellular carcinoma.   (View pathway)

· Human Diseases > Cancers: Specific types > Gastric cancer.   (View pathway)

· Human Diseases > Immune diseases > Inflammatory bowel disease (IBD).

· Organismal Systems > Immune system > Th17 cell differentiation.   (View pathway)

· Organismal Systems > Endocrine system > Relaxin signaling pathway.

References

1). Liu H et al. Enhancer of zeste homolog 2 modulates oxidative stress-mediated pyroptosis in vitro and in a mouse kidney ischemia-reperfusion injury model. FASEB J 2020 Jan;34(1):835-852 (PubMed: 31914694) [IF=4.966]

Application: WB    Species: human    Sample: HK-2 cells

FIGURE 7|EZH2 regulated Nox4 expression via the ALK5/Smad2/Smad3 pathway. D-G, HK-2 cells were transfected with an siRNA against EZH2 or a negative control siRNA (si-NC) for 48 h before being exposed to H/R. D-F, Western blot analysis for the protein expression of ALK5, Smad2, Smad3, p-Smad2, and Smad3 in the indicated groups and quantitative analysis of ALK5, p-Smad2, and p-Smad3, n = 3.

2). Li X et al. Myricetin ameliorates bleomycin-induced pulmonary fibrosis in mice by inhibiting TGF-β signaling via targeting HSP90β. Biochem Pharmacol 2020 Jun 11;114097. (PubMed: 32535102) [IF=4.960]

3). Li X et al. Regorafenib-Attenuated, Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway. Int J Mol Sci 2021 Feb 17;22(4):1985. (PubMed: 33671452) [IF=4.556]

Application: WB    Species: Mouse    Sample: Mlg cells

Figure 3. Regorafenib down-regulates TGF-β1/Smad and TGF-β1/non-Smad signals in pulmonary fibroblasts. (A) CAGAmouse embryonic fibroblast (NIH-3T3) cells were exposed to TGF-β1 (5 ng/mL) or a series concentration (0–32 µM) in serum-free medium for 18 h; (B) Mlg cells were treated with TGF-β1 (5 ng/mL) and/or RG (2 µM, 4 µM) for 30 min, and Western blot was used to detect Smad3, Smad2, and their phosphorylation expression levels. Densitometric analysis are shown beside; (C) Mlg cells were incubated with RG (2 µM, 4 µM) and/or TGF-β1 (5 ng/mL) for 12 h to analyze the Erk1/2 and Akt and their phosphorylation levels by Western blotting. Densitometric analysis are shown beside; (D) BLM-PPF cells were incubated with RG (2 µM, 4 µM) for 12 h to analyze Erk1/2 and Akt and their phosphorylation levels by Western blotting. Densitometric analysis are shown beside. β-tubulin or GAPDH were used as a loading control. Data in (A–D) are means ± standard error of mean (SEM); ### p < 0.001, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA), NS: not significant.

4). Wei J et al. Mesenchymal stem cells ameliorate silica‐induced pulmonary fibrosis by inhibition of inflammation and epithelial‐mesenchymal transition. J Cell Mol Med 2021 Jun 2. (PubMed: 34076355) [IF=4.486]

Application: WB    Species: rat    Sample: lung

FIGURE 5|BMSCs blocked the activation of TGF-β/Smad pathway. (D) Western blot results of TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3 and Smad7 protein expression levels. n = 3 rats per group.

5). Chen G et al. Myricetin suppresses the proliferation and migration of vascular smooth muscle cells and inhibits neointimal hyperplasia via suppressing TGFBR1 signaling pathways. Phytomedicine 2021 Nov;92:153719. (PubMed: 34500301) [IF=4.268]

6). Jiang Y et al. Transcriptomic analysis of the mechanisms of alleviating renal interstitial fibrosis using the traditional Chinese medicine Kangxianling in a rat model. Sci Rep 2020 Jun 30;10(1):10682. (PubMed: 32606425) [IF=3.998]

Application: WB    Species: rat    Sample: renal

Figure 2. |Marker validation in the RIF model. (a) Te expression levels of the renal fbrosis-associated genes TGF-β1, FN, Smad3, Col1a1, Col1a2, and α-SMA were quantitated in each of the groups. All groups were compared with OC. *stands for p<0.05, **stands for p<0.01, and ***stands for p<0.001. (b) Te western blot analysis of TGF-β1, Smad3, a-SMA, FN, Col1a1 and Col1a2.

7). Tan Z et al. Taohong siwu decoction attenuates myocardial fibrosis by inhibiting fibrosis proliferation and collagen deposition via TGFBR1 signaling pathway. J Ethnopharmacol 2021 Jan 16;270:113838. (PubMed: 33460756) [IF=3.690]

Application: WB    Species: mice    Sample: cardiac fibroblasts

Fig. 6. THSWD suppresses expression of collagen and activation of the TGFBR1 signaling pathway. (A, B) CFs were incubated without or with TGF-β1 (10 ng/ml) and THSWD (15, 30 and 60 μg/ml) for 24 h, and the expression levels of collagen I, collagen III, collagen V, phospho-TGFBR1, TGFBR1, phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were tested by western blotting. (C–E) Expression levels of collagen I, collagen III and collagen V were normalized with GAPDH (n = 3). (F–H) Expression levels of phospho-TGFBR1, phospho-Smad2, and phospho-Smad3 were normalized to that of TGFBR1, Smad2 and Smad3 proteins, respectively (n = 3). Data were shown as mean ± SD. #P < 0.05, vs. control group. *P < 0.05, **P < 0.01, vs. model group.

Application: IHC    Species: mice    Sample: heart tissues

Fig. 4. IHC analysis of TGFBR1 signaling pathway related-protein expression in heart tissues. (A) IHC showed inhibition of TGFBR1, Smad3, collagen I, collagen III and α-SMA in THSWD-treated mouse heart tissues compared with the model group. (B–F) Quantitative analysis for IHC staining of TGFBR1, Smad3, collagen I, collagen III and α-SMA. Data were shown as mean ± SD. **P < 0.01, vs. model group.

8). Ren W et al. Arginine inhibits the malignant transformation induced by interferon-gamma through the NF-κB-GCN2/eIF2α signaling pathway in mammary epithelial cells in vitro and in vivo. Exp Cell Res 2018 Jul 15;368(2):236-247 (PubMed: 29746817) [IF=3.383]

Application: WB    Species: mouse    Sample: primary cells and bovine mammary epithelial cells

Fig. 3.| Differentially expressed genes associated with cancer in transcriptomics.(C) Representative proteins were investigated using western blot analysis. The cells of two group were treated for twenty-four hours. In Fig. B and C, NC/0: primary cells, N30/30: thirty generations of bovine mammary epithelial cells treated by IFN-γ (10 ng/ml). RNAseq: transcriptomics results. qRT-PCR: fluorescence quantitative PCR results. The data represent the means ± SEM of 3 independent experiments. Each bar represents the mean of three independent experiments. One-way ANOVA; **P < 0.01.

9). Ruan H et al. Deglycosylated Azithromycin Attenuates Bleomycin-Induced Pulmonary Fibrosis via the TGF-β1 Signaling Pathway. Molecules 2021 May 10;26(9):2820. (PubMed: 34068694) [IF=3.267]

Application: WB    Species: mouse    Sample: lung

Figure 7.| Deglycosylated azithromycin inhibits the fibrogenic activation of pulmonary fibroblasts in vivo.(K) Western blot analysis of the protein levels of p-Akt, p-Smad3, p-p38 and p-Erk1/2 expression.

10). Wang F et al. High-dose vitamin D3 c ameliorates renal fibrosis by vitamin D receptor activation and inhibiting TGF-β1/Smad3 signaling pathway in 5/6 nephrectomized rats. Eur J Pharmacol 2021 Jun 17;907:174271. (PubMed: 34147475) [IF=3.263]

Application: IHC    Species: Rat    Sample: kidney tissues

Fig. 6. Vitamin D3 activated the vitamin D receptor and inhibited the activation of the TGF-β1/Smad3 signaling pathway. Renal expression of (A) Vitamin D receptor, (B) TGF-β1, and (C) Smad3 mRNA was determined by RT-qPCR. (D) Protein expression levels of the vitamin D receptor and TGF-β1 analyzed by Western blot; (E, F) Vitamin D receptor and TGF-β1 protein expression relative to GADPH protein expression; (G) p-Smad3 protein expression visualized in renal sections using immunohistochemistry (scale bar: 100 μm); (H) average optical density (AOD) analysis of p-Smad3. Data expressed as mean ± S.E.M. (n = 3–7), *P < 0.05 vs sham group; **P < 0.01 vs sham group; ***P < 0.001 vs sham group; #P < 0.05 vs 5/6 Nx group; ##P < 0.01 vs 5/6 Nx group; ###P < 0.001 vs 5/6 Nx group; ns, no significance.

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