Overview

  • Product nameAnti-Smad3 antibody [AF9F7]See all Smad3 primary antibodies ...
  • Description
    Mouse monoclonal [AF9F7] to Smad3
  • Tested applicationsIP, Flow Cyt, Sandwich ELISA, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Ferret, Zebrafish
  • Immunogen

    Recombinant full length Smad3 protein (Human) purified from E.coli (His-Smad3)

  • Positive control
    • Hela, IMR32, A431, Jurkat and PC12 cell lysates

Properties

Applications

Our Abpromise guarantee covers the use of ab75512 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
IP Use a concentration of 5 µg/ml.
Flow Cyt 1/100.
Sandwich ELISA Use a concentration of 5 µg/ml. Can be paired for Sandwich ELISA with Rabbit monoclonal [EP568Y] to Smad3 (ab40854). For sandwich ELISA, use this antibody as Capture at 5 µg/ml with Rabbit monoclonal [EP568Y] to Smad3 (ab40854) as Detection.
WB 1/5000. Detects a band of approximately 56 kDa (predicted molecular weight: 48 kDa).
ELISA Use at an assay dependent dilution.

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 antibody [AF9F7] images

  • Standard Curve for Smad3 (Analyte: Smad3 protein (His tag) (ab89353)); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [AF9F7] to Smad3 (ab75512) at 5µg/ml and Detector Antibody Rabbit monoclonal [EP568Y] to Smad3 (ab40854) at 0.5µg/ml.
  • All lanes : Anti-Smad3 antibody [AF9F7] (ab75512) at 1/5000 dilution

    Lane 1 : Hela cell lysate
    Lane 2 : IMR32 cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : Jurkat cell lysate
    Lane 5 : PC12 cell lysate


    Predicted band size : 48 kDa
    Observed band size : 56 kDa (why is the actual band size different from the predicted?)
  • Overlay histogram showing HCT116 cells stained with ab75512 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75512, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Smad3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal 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 ab75512.
    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
    Band: 56kDa; Smad3

Protocols

References for Anti-Smad3 antibody [AF9F7] (ab75512)

This product has been referenced in:
  • Nolan-Stevaux O  et al. Endoglin requirement for BMP9 signaling in endothelial cells reveals new mechanism of action for selective anti-endoglin antibodies. PLoS One 7:e50920 (2012). WB ; Rat . Read more (PubMed: 23300529) »

See 1 Publication for this product

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I have been in contact with the lab about the issues you have been having with ab75512 and I attached the test result for cross reactivity with Smad families. However, we’ve not tested with smad3 KO mouse.
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The antibody, ab75512, is covered under our Abpromise for six months and is guaranteed to work in western blot on mouse samples . If we cannot resolve the issue you are having with the antibody then I would be ...

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I received a reply from the lab about the concentration of ab75512. They informed me that they have not measure the actual concentration of this antibody. They have estimated that the concentration is between 0.5-2mg/ml. I am sorry not to be able to p...

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The product ab75512 has been tested for cross-reactivity against recombinant proteins from Smad1 to 5. As a result, it has been shown that this product is specific to Smad3 as by western blotting there was only reaction with Smad3.

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