Recombinant
RabMAb

Anti-Smad3 antibody [EP568Y] (ab40854)

Overview

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 Abreviews Notes
WB 1/1000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa).Can be blocked with Smad3 peptide (ab173094).

 

IHC-P 1/500 - 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

IF Use at an assay dependent concentration. PubMed: 28135282
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 1/50 - 1/210.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/500 - 1/2000.

 

  • Application notes
    Is unsuitable for IP.
  • Target

    • 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.
    • Involvement in disease
      Colorectal cancer
      Loeys-Dietz syndrome 3
    • Sequence similarities
      Belongs to the dwarfin/SMAD family.
      Contains 1 MH1 (MAD homology 1) domain.
      Contains 1 MH2 (MAD homology 2) domain.
    • Domain
      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.
    • 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.
      Poly-ADP-ribosylated by PARP1 and PARP2. ADP-ribosylation negatively regulates SMAD3 transcriptional responses during the course of TGF-beta signaling.
    • Cellular localization
      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).
    • 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 [EP568Y] images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labelling unpurified ab40854.

    • All lanes : Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution (purified)

      Lane 1 : HT-29 cell lysate
      Lane 2 : HT-1080 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size : 48 kDa
      Observed band size : 58 kDa (why is the actual band size different from the predicted?)

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Immunocytochemsitry/Immunofluorescence analysis of HepG2 cells labelling Smad3 (green) with purified ab40854 at 1/2000. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

    • Overlay histogram showing HCT116 cells stained with unpurified 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.

    • Standard Curve for Smad3 (Analyte: Smad3 protein (His tag) (ab89353, unpurified)); 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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Smad3 with purified ab40854 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

    • Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution (purified) + Rat liver tissue lysate at 10 µg

      Secondary
      Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size : 48 kDa
      Observed band size : 58 kDa (why is the actual band size different from the predicted?)

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Flow cytometry analysis of HT-29 cells labelling Smad3 with purified ab40854 at 1/210 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.

    • ab40854 staining Smad3 in human granulosa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with ethanol and triton and blocked for 1 hour at 26°C. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An undiluted IRDye® 800CW-conjugated goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody. Left - negative control (4 replicates).

      See Abreview

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate carcinoma tissue labelling unpurified ab40854 at 1/100 dilution.

    • Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5000 dilution (unpurified) + 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?)
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling unpurified ab40854.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarcinoma tissue labelling unpurified ab40854.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue labelling unpurified ab40854.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labelling unpurified ab40854.

    Protocols

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

    This product has been referenced in:

    See all 36 Publications for this product

    Product Wall

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Lung tissue)
    Antigen retrieval step
    None
    Permeabilization
    No
    Specification
    Lung tissue
    Blocking step
    Zytomed Systems HRP Polymer Blocking Reagent as blocking agent for 5 minute(s) · Concentration: 100% · Temperature: 25°C
    Fixative
    HOPE-fixation
    Username

    Abcam user community

    Verified customer

    Submitted Oct 06 2015

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Saos)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    20 µg
    Specification
    Saos
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5µg/mL · Temperature: 21°C
    Username

    Abcam user community

    Verified customer

    Submitted Apr 21 2015

    Application
    Western blot
    Loading amount
    20 µg
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Human Cell lysate - whole cell (Normal Human Keratinocyte)
    Specification
    Normal Human Keratinocyte
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Username

    Abcam user community

    Verified customer

    Submitted Mar 30 2015

    Application
    Western blot
    Loading amount
    50 µg
    Gel Running Conditions
    Reduced Denaturing (4-12%)
    Sample
    Human Cell lysate - whole cell (HeLa)
    Specification
    HeLa
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Username

    Abcam user community

    Verified customer

    Submitted Oct 24 2014

    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
    Sample
    Human Cell (Human granulosa cell Line)
    Specification
    Human granulosa cell Line
    Permeabilization
    Yes - Ethanol and triton
    Fixative
    Paraformaldehyde
    Username

    Dr. Francesco Elia Marino

    Verified customer

    Submitted Jun 09 2014

    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

    Dr. 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.

    Abcam guarantees this product to work in the species/application used in this Abreview.
    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"

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