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  • Product Name
    Phospho-Smad3 (Ser425) Antibody
  • Catalog No.
    AF3362
  • RRID
    AB_2834777
  • Source
    Rabbit
  • Application
    WB,IHC,IF/ICC,ELISA(peptide)
  • Reactivity
    Human, Mouse, Rat
  • Prediction
    Pig, Bovine, Horse, Sheep, Rabbit, Dog, Chicken, Xenopus
  • UniProt
  • Mol.Wt
    58kD;
    48kDa(Calculated).
  • Concentration
    1mg/ml
  • Browse similar products>>

Related Products

Product Information

Alternative Names:Expand▼

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;

Applications:

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

Predicted Reactivity:

Pig, Bovine, Horse, Sheep, Rabbit, Dog, Chicken, Xenopus

Source:

Rabbit

Clonality:

Polyclonal

Purification:

The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.

Specificity:

Phospho-Smad3 (Ser425) Antibody detects endogenous levels of Smad3 only when phosphorylated at Serine 425.

RRID:

AB_2834777
Please cite this product as: Affinity Biosciences Cat# AF3362, RRID:AB_2834777.

Format:

Liquid

Concentration:

1mg/ml

Storage Condition and Buffer:

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.

Immunogen Information in 3D

Immunogen:

A synthesized peptide derived from human Smad3 around the phosphorylation site of Ser425.

Uniprot:



>>Visit The Human Protein Atlas

Gene ID:

Gene Name:

SMAD3

Molecular Weight:

Observed Mol.Wt.: 58kD.
Predicted Mol.Wt.: 48kDa(Calculated)..

Subcellular Location:

Cytoplasm. Nucleus. 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).

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

Research Background

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.

Post-translational Modifications:

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 assertionGraphics by Christian Stolte

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.

Similarity:

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

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.

Reference Citations:

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]

2). Li X;Yu H;Liang L;Bi Z;Wang Y;Gao S;Wang M;Li H;Miao Y;Deng R;Ma L;Luan J;Li S;Liu M;Lin J;Zhou H;Yang C; 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). Wei J;Zhao Q;Yang G;Huang R;Li C;Qi Y;Hao C;Yao W; 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=4.658]

4). Wang S et al. Roscovitine attenuates renal interstitial fibrosis in diabetic mice through the TGF-β1/p38 MAPK pathway. Biomed Pharmacother 2019 Apr 24;115:108895 (PubMed: 31029000) [IF=4.545]

5). Wei J;Zhao Q;Yang G;Huang R;Li C;Qi Y;Hao C;Yao W; 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]

6). Li X;Liu R;Cui Y;Liang J;Bi Z;Li S;Miao Y;Zhang L;Li X;Zhou H;Yang C; et al. Protective effect of remdesivir against pulmonary fibrosis in mice. Front Pharmacol 2021 Aug 26;12:692346. (PubMed: 34512328) [IF=4.225]

7). Wang XF;Fang QQ;Jia B;Hu YY;Wang ZC;Yan KP;Yin SY;Liu Z;Tan WQ; et al. Potential Effect of Non-Thermal Plasma for the Inhibition of Scar Formation: A Preliminary Report. Sci Rep 2020 Jan 23;10(1):1064. (PubMed: 31974451) [IF=3.998]

8). Gu L et al. Ribes diacanthum Pall (RDP) ameliorates UUO-induced renal fibrosis via both canonical and non-canonical TGF-β signaling pathways in mice. J Ethnopharmacol 2018 Oct 17 (PubMed: 30342194) [IF=3.690]

9). Liu YL;Yang WH;Chen BY;Nie J;Su ZR;Zheng JN;Gong ST;Chen JN;Jiang D;Li Y; et al. miR‑29b suppresses proliferation and induces apoptosis of hepatocellular carcinoma ascites H22 cells via regulating TGF‑β1 and p53 signaling pathway. Int J Mol Med 2021 Aug;48(2):157. (PubMed: 34184070) [IF=3.098]

10). Zhao J et al. TGF-β1 and connexin-43 expression in neurogenic bladder from rats with sacral spinal cord injury. Neurourol Urodyn 2018 Aug 2 (PubMed: 30070388)

11). Sun Y;Lin J;Luo Z;Zhang Y;Chen J; et al. The Serum from Patients with Secondary Frozen Shoulder Following Rotator Cuff Repair Induces Shoulder Capsule Fibrosis and Promotes Macrophage Polarization and Fibroblast Activation. J Inflamm Res 2021 Mar 23;14:1055-1068. (PubMed: 33790620)

12). et al. Zanubrutinib Attenuated Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway.

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Catalog Number :

AF3362-BP
(Blocking peptide available as AF3362-BP)

Price/Size :

$350/1mg.
Tips: For phospho antibody, we provide phospho peptide(0.5mg) and non-phospho peptide(0.5mg).

Function :

Blocking peptides are peptides that bind specifically to the target antibody and block antibody binding. These peptide usually contains the epitope recognized by the antibody. Antibodies bound to the blocking peptide no longer bind to the epitope on the target protein. This mechanism is useful when non-specific binding is an issue, for example, in Western blotting (immunoblot) and immunohistochemistry (IHC). By comparing the staining from the blocked antibody versus the antibody alone, one can see which staining is specific; Specific binding will be absent from the western blot or immunostaining performed with the neutralized antibody.

Format and storage :

Synthetic peptide was lyophilized with 100% acetonitrile and is supplied as a powder. Reconstitute with 0.1 ml DI water for a final concentration of 10 mg/ml.The purity is >90%,tested by HPLC and MS.Storage Maintain refrigerated at 2-8°C for up to 6 months. For long term storage store at -20°C.

Precautions :

This product is for research use only. Not for use in diagnostic or therapeutic procedures.

Pig
100%
Horse
100%
Bovine
100%
Sheep
100%
Dog
100%
Xenopus
100%
Chicken
100%
Rabbit
100%
Zebrafish
0%
High similarity Medium similarity Low similarity No similarity
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)
IMPORTANT: For western blots, incubate membrane with diluted antibody in 5% w/v milk , 1X TBS, 0.1% Tween®20 at 4°C with gentle shaking, overnight.

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