RabMAb

Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

Overview

  • Product nameAnti-Smad3 (phospho S423 + S425) antibody [EP823Y]
    See all Smad3 primary antibodies
  • Description
    Rabbit monoclonal [EP823Y] to Smad3 (phospho S423 + S425)
  • Specificityab52903 detects Smad3 phosphorylated on Serine 423 and Serine 425. This Smad3 antibody may also detect Smad1, Smad2 and Smad5 phosphorylated at the equivalent sites.
  • Tested applicationsWB, ICC/IF, ELISA, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human

    Does not react with

    Rat
  • Immunogen

    A synthetic phospho specific peptide corresponding to residues surrounding Ser423 and Ser425 of human Smad3.

  • Positive control
    • WB: HL-60 cell lysates IHC-P: Human liver carcinoma ICC/IF: HeLa cells
  • General notes

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

Properties

Applications

Our Abpromise guarantee covers the use of ab52903 in the following tested applications.

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

Application Abreviews Notes
WB 1/2000. Predicted molecular weight: 48 kDa.
ICC/IF 1/100 - 1/250.
ELISA Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration. PubMed: 23667656
IHC-P 1/100 - 1/250.
  • Application notesIs unsuitable for Flow Cyt or IP.
  • 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. 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
      Loeys-Dietz syndrome 3
    • 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.
    • Information by UniProt
    • Database links
    • Alternative names
      • DKFZP586N0721 antibody
      • DKFZp686J10186 antibody
      • hMAD 3 antibody
      • hMAD-3 antibody
      • hSMAD3 antibody
      • HSPC193 antibody
      • HST17436 antibody
      • JV15 2 antibody
      • JV15-2 antibody
      • JV152 antibody
      • LDS1C antibody
      • LDS3 antibody
      • MAD (mothers against decapentaplegic Drosophila) homolog 3 antibody
      • MAD homolog 3 antibody
      • Mad homolog JV15 2 antibody
      • Mad protein homolog antibody
      • MAD, mothers against decapentaplegic homolog 3 antibody
      • MAD3 antibody
      • MADH 3 antibody
      • MADH3 antibody
      • MGC60396 antibody
      • Mothers against decapentaplegic homolog 3 antibody
      • Mothers against DPP homolog 3 antibody
      • SMA and MAD related protein 3 antibody
      • SMAD 3 antibody
      • SMAD antibody
      • SMAD family member 3 antibody
      • SMAD, mothers against DPP homolog 3 antibody
      • SMAD3 antibody
      • SMAD3_HUMAN antibody
      see all

    Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] images

    • ab52903 staining Smad3 in mouse primary embryonic epicardial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and blocked with PBS + 1% BSA + 10% goat serum + 0.1% Triton X-100 for 1 hour at 20°C. Samples were incubated with primary antibody (1/100 in PBS + 1% BSA + 10% goat serum + 0.1% Triton X-100) for 16 hours at 4°C. An Alexa Fluor®488-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

      See Abreview



    • Predicted band size : 48 kDa
    • All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/2000 dilution

      Lane 1 : (A) HL-60 cell lysates at 10µg untreated
      Lane 2 : (B) HL-60 cell lysates at 10µg treated with TGF.


      Predicted band size : 48 kDa
      Observed band size : 55 kDa (why is the actual band size different from the predicted?)
      Additional bands at : 45 kDa. We are unsure as to the identity of these extra bands.
    • Immunofluorescent staining of Smad3 in HeLa cells using ab52903 at 1/100 dilution.
    • Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.
    • All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution

      Lane 1 : Lysate prepared from untreated human A549 cells
      Lane 2 : Lysate prepared from untreated human A549 cells for 30min
      Lane 3 : Lysate prepared from TGF-ß1 cells at 10ng/ml for 30min
      Lane 4 : Lysate prepared from TNF-a cells at 20ng/ml for 30min
      Lane 5 : Lysate prepared from TGF-ß1 and TNF-a cells at above doses for 30min
      Lane 6 : Blank DMEM media

      Lysates/proteins at 20 µg per lane.

      Secondary
      Donkey Anti-Rabbit IgG H&L (HRP) (ab16284)
      developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 48 kDa
      Observed band size : 48 kDa


      Exposure time : 1 hour

      This image is a courtesy of Aaron Gardner

      See Abreview

    • ab52903 staining Smad3 (phospho S423 + S425) in human TII Pneumocyte A549c ells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton x100 before blocking with 3% BSA for 1 hour at RT. Samples were incubated with primary antibody (1/200: in 3% BSA in 1x PBST) for 24 hours at 4°C. A TRITC-conjugated goat polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution.

      See Abreview

    References for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

    This product has been referenced in:
    • Sulkowski M  et al. Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction. Development 141:436-47 (2014). Read more (PubMed: 24353060) »
    • Savar A  et al. Loss of epithelial p53 and av integrin cooperate through Akt to induce squamous cell carcinoma yet prevent remodeling of the tumor microenvironment. Oncogene N/A:N/A (2014). WB ; Mouse . Read more (PubMed: 24469034) »

    See all 33 Publications for this product

    Product Wall

    Application Immunocytochemistry/ Immunofluorescence
    Blocking step PBS, 1% BSA, 10% goat serum, 0.1% TX-100 as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
    Sample Mouse Cell (primary embryonic epicardial)
    Specification primary embryonic epicardial
    Permeabilization Yes - 0.5% Triton X-100
    Fixative Formaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted Jan 30 2014

    Application Western blot
    Loading amount 60 µg
    Gel Running Conditions Reduced Non-Denaturing (Native) (12)
    Sample Human Tissue lysate - whole (Human Testis)
    Specification Human Testis
    Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
    Username

    Mr. FRANCESCO ELIA MARINO

    Verified customer

    Submitted Mar 12 2014

    Thank you for contacting us and your interest in our products.

    The protocol used with the anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) to stain formalin fixed, paraffin embedded tissue sections is as attached to this email. Read More

    Thank you for your inquiry.
    We added the blocking peptide for ab52903 to our catalog under the product number ab135224.
    Please follow this link to review the datasheet:
    http://www.abcam.com/index.html?datasheet=135224 http://www.abcam.co...

    Read More

    Thank you for your reply.

    I have processed your request to receive a new vial of ab52903 since the current vial you have has not been working for you. Since you purchased it through Cederlane, then we will have to ship the antibody to them fir...

    Read More

    Thank you for the reply.

    The only order I am finding from the University of Western Ontario is from March 2011, but the PO# is different. Do you order directly from Abcam, or do you go through one of our distributors? Also, roughly when was th...

    Read More

    Thank you for that information.

    I just have one more question, sorry meant to ask it in the previous email, which institution are you with? I just want to make sure that I locate the correct order and send the replacement to the correct person...

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    Thank you for your reply.

    I am sorry that using the BSA did not prove to be effective and I agree that there may be something wrong with the vial of antibody that you have. Therefore I would be more than happy to either replace or refund that ...

    Read More

    Thank you for contacting Abcam.

    The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on mouse and human samples. If we cannot resolve the issue you are having with the antibody then I would be h...

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (HEK293)
    Loading amount 15 µg
    Specification HEK293
    Treatment Various DMSO concs over 48hrs
    Gel Running Conditions Non-reduced Denaturing (10% gel)
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Username

    Abcam user community

    Verified customer

    Submitted May 17 2012

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