Overview

  • Product nameAnti-Smad3 antibody [EP568Y]See all Smad3 primary antibodies ...
  • Description
    Rabbit monoclonal [EP568Y] to Smad3
  • SpecificityThis antibody is specific for Smad3.
  • Tested applicationsWB, ICC, IHC-P, Sandwich ELISA, Flow Cyt more details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to the internal region of Human Smad3.

  • Positive control
    • Jurkat cell lysate and human prostate carcinoma tissue.
  • General notesProduced under U.S. Patent No. 5,675,063.

Properties

Applications

Our Abpromise guarantee covers the use of ab40854 in the following tested applications.

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

Application Notes
WB 1/5000. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa).Can be blocked with Smad3 peptide (ab173094).
ICC 1/100 - 1/250.
IHC-P Use at an assay dependent concentration.
Sandwich ELISA Use a concentration of 0.5 µg/ml. Can be paired for Sandwich ELISA with Mouse monoclonal [AF9F7] to Smad3 (ab75512). For sandwich ELISA, use this antibody as Detection at 0.5 µg/ml with Mouse monoclonal [AF9F7] to Smad3 (ab75512) as Capture.
Flow Cyt 1/100.
  • Application notesIs unsuitable for 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.
    • 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
    • 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
      • JV152 antibody
      • LDS1C antibody
      • LDS3 antibody
      • MAD (mothers against decapentaplegic Drosophila) homolog 3 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
      • MAD3 antibody
      • MADH 3 antibody
      • MADH3 antibody
      • MADH3 antibody
      • MGC60396 antibody
      • Mothers against decapentaplegic homolog 3 antibody
      • Mothers against DPP 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 [EP568Y] 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.
    • Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5000 dilution + Jurkat cell lysate at 10 µg

      Predicted band size : 48 kDa
      Observed band size : 55 kDa (why is the actual band size different from the predicted?)
    • ab40854 at a 1:100 dilution staining Smad3 in human prostate carcinoma tissue.
    • ab40854 showing positive staining in Breast carcinoma tissue.

    • ab40854 showing positive staining in Colonic adenocarcinoma tissue.

    • ab40854 showing positive staining in Gastric adenocarcinoma tissue.

    • ab40854 showing positive staining in Glioma tissue.

    • ab40854 showing positive staining in Lung adenocarcinoma tissue.

    • Overlay histogram showing HCT116 cells stained with ab40854 (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 (ab40854, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    Protocols

    References for Anti-Smad3 antibody [EP568Y] (ab40854)

    This product has been referenced in:
    • Wei X  et al. Kindlin-2 Mediates Activation of TGF-ß/Smad Signaling and Renal Fibrosis. J Am Soc Nephrol 24:1387-98 (2013). Human . Read more (PubMed: 23723426) »
    • Nakajima T  et al. Temporal and regional patterns of Smad activation in the rat hippocampus following global ischemia. J Neurol Sci N/A:N/A (2013). WB, IHC ; Rat . Read more (PubMed: 24290497) »

    See all 8 Publications for this product

    Product Wall

    Application Western blot
    Loading amount 40 µg
    Gel Running Conditions Non-reduced Non-Denaturing (Native) (12)
    Sample Mouse Tissue lysate - whole (Gastrocnemius (Muscle))
    Specification Gastrocnemius (Muscle)
    Blocking step Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 27°C
    Username

    Mr. FRANCESCO ELIA MARINO

    Verified customer

    Submitted Mar 07 2014

    Thank you for contacting us.

    We do have a number of anti-SMAD2 and anti-SMAD3 antibodies which we guarantee in IHC-P on mouse. We to don't have the opportunity to test specific products against one another in each application but based on ...

    Read More

    Thank you for contacting Abcam.
    For ab40854, an immunogen that is located between amino acids ******and******* was used.
    If there is anything else I can help you with, please let me know.

    Application Western blot
    Sample Human Cell lysate - whole cell (Hela Cells)
    Loading amount 30 µg
    Specification Hela Cells
    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 Jun 13 2011

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