You have changed your country from  to  . Please be aware that this will change the currency in the purchasing process.

Overview

  • Product nameAnti-Smad3 antibodySee all Smad3 primary antibodies ...
  • Description
    Rabbit polyclonal to Smad3
  • Tested applicationsIP, ICC/IF, WB, IHC-P more details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat, Sheep, Horse, Chicken, Pig
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 150 - 250 of Human Smad3.

    Read Abcam's proprietary immunogen policy

    (Peptide available as ab95046.)

  • Positive controlThis antibody gave a positive signal in HeLa whole cell lysate; HeLa nuclear extract and EGF-treated A431 whole cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab84177 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IP IP: Use at an assay dependent concentration.
ICC/IF ICC/IF: Use a concentration of 5 µg/ml.
WB WB: Use a concentration of 1 µg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 48 kDa).Can be blocked with Smad3 peptide (ab95046).
IHC-P IHC-P: Use a concentration of 1 µg/ml.

Target

  • FunctionReceptor-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 (By similarity). Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.
  • Involvement in diseaseColorectal cancer (CRC) [MIM:114500]: A complex disease characterized by malignant lesions arising from the inner wall of the large intestine (the colon) and the rectum. Genetic alterations are often associated with progression from premalignant lesion (adenoma) to invasive adenocarcinoma. Risk factors for cancer of the colon and rectum include colon polyps, long-standing ulcerative colitis, and genetic family history. Note=The disease may be caused by mutations affecting the gene represented in this entry.
    Loeys-Dietz syndrome 3 (LDS3) [MIM:613795]: An aortic aneurysm syndrome with widespread systemic involvement. The disorder is characterized by the triad of arterial tortuosity and aneurysms, hypertelorism, and bifid uvula or cleft palate. Patients with LDS3 also manifest early-onset osteoarthritis. They lack craniosynostosis and mental retardation. Note=The disease is caused by mutations affecting the gene represented in this entry. SMAD3 mutations have been reported to be also associated with thoracic aortic aneurysms and dissection (TAAD) (PubMed:21778426). This phenotype is distinguised from LDS3 by having aneurysms restricted to thoracic aorta. As individuals carrying these mutations also exhibit aneurysms of other arteries, including abdominal aorta, iliac, and/or intracranial arteries (PubMed:21778426), they have been classified as LDS3 by the OMIM resource.
  • Sequence similaritiesBelongs to the dwarfin/SMAD family.
    Contains 1 MH1 (MAD homology 1) domain.
    Contains 1 MH2 (MAD homology 2) domain.
  • DomainThe 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.
  • 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.
    Ubiquitinated. Monoubiquitinated, leading to prevent DNA-binding. Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes.
  • Cellular localizationCytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. Co-localizes with LEMD3 at the nucleus inner membrane. MAPK-mediated phosphorylation appears to have no effect on nuclear import. PDPK1 prevents its nuclear translocation in response to TGF-beta.

    • Target information above from: UniProt accession P84022 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links
    see all
  • Alternative names
      DKFZP586N0721 antibodyDKFZp686J10186 antibodyhMAD 3 antibody
      hMAD-3 antibodyhSMAD3 antibodyHSPC193 antibodyHST17436 antibodyJV15 2 antibodyJV15-2 antibodyJV152 antibodyLDS1C antibodyLDS3 antibodyMAD (mothers against decapentaplegic Drosophila) homolog 3 antibodyMAD homolog 3 antibodyMad homolog JV15 2 antibodyMad protein homolog antibodyMAD, mothers against decapentaplegic homolog 3 antibodyMAD3 antibodyMADH 3 antibodyMADH3 antibodyMGC60396 antibodyMothers against decapentaplegic homolog 3 antibodyMothers against DPP homolog 3 antibodySMA and MAD related protein 3 antibodySMAD 3 antibodySMAD antibodySMAD family member 3 antibodySMAD, mothers against DPP homolog 3 antibodySMAD3 antibodySMAD3_HUMAN antibody
    see all

Anti-Smad3 antibody images

  • Western blot - Smad3 antibody (ab84177)
    Western blot - Smad3 antibody (ab84177)
    All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml

    Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : EGF-treated A431 (Human epithelial carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg/ml per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 48 kDa
    Observed band size : 52 kDa (why is the actual band size different from the predicted?)


    Exposure time : 20 minutes
  • Immunoprecipitation - Anti-Smad3 antibody (ab84177)
    Immunoprecipitation - Anti-Smad3 antibody (ab84177)
    Smad3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Smad3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab84177.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 50kDa: Smad3
  • Immunocytochemistry/ Immunofluorescence - Smad3 antibody (ab84177)
    Immunocytochemistry/ Immunofluorescence - Smad3 antibody (ab84177)
    ICC/IF image of ab84177 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab84177, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa cells at 5µg/ml.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Smad3 antibody (ab84177)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Smad3 antibody (ab84177)
    IHC image of Ab84177 staining in Human Cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with Ab84177, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • Western blot - Smad3 antibody (ab84177)
    Western blot - Smad3 antibody (ab84177)
    All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 48 kDa
    Observed band size : 48 kDa
    Additional bands at : 37 kDa,50 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 8 minutes

References for Anti-Smad3 antibody (ab84177)

ab84177 has not yet been referenced specifically in any publications.

Publishing research using ab84177 ? Please let us know so that we can cite the reference in this datasheet

Product Wall

Submit an Abreview for ab84177 Submit a Question for ab84177

There are currently no Abreviews or Questions for ab84177.
Please use the links above to contact us or submit feedback about this product.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"