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RabMAb

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

製品の概要

  • 製品名
    Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]
    Smad3 一次抗体 製品一覧
  • 製品の詳細
    Rabbit monoclonal [EP823Y] to Smad3 (phospho S423 + S425)
  • 特異性
    ab52903 detects Smad3 phosphorylated on Serine 423 and Serine 425. This Smad3 antibody may also detect Smad1, Smad2 and Smad5 phosphorylated at the equivalent sites.
  • アプリケーション
    適用あり: WB, ICC/IF, ELISA, IHC-Fr, IHC-Pmore details
    適用なし: Flow Cyt or IP
  • 種交差性
    交差種: Mouse, Human, Drosophila melanogaster
  • 免疫原

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

  • ポジティブ・コントロール
    • WB: HL-60 treated with TGF cell lysates. IHC-P: Human liver carcinoma tissue. ICC/IF: TGFß treated A549 cells.
  • 特記事項

    This product is a recombinant rabbit monoclonal antibody.

     

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
WB 1/2000. Predicted molecular weight: 48 kDa.

Avoid using milk, casein, and phopshorylated proteins in general in the blocking buffer and in the antibody diluent. We recommend a solution of 5% BSA (bovine serum albumin).

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.

The secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (ab209101)

IF Use at an assay dependent concentration. PubMed: 28135282
  • 追加情報
    Is unsuitable for Flow Cyt or IP.
  • ターゲット情報

    • 機能
      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.
    • 関連疾患
      Colorectal cancer
      Loeys-Dietz syndrome 3
    • 配列類似性
      Belongs to the dwarfin/SMAD family.
      Contains 1 MH1 (MAD homology 1) domain.
      Contains 1 MH2 (MAD homology 2) domain.
    • ドメイン
      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.
    • 翻訳後修飾
      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.
    • 細胞内局在
      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
    • 参照データベース
    • 別名
      • 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

    画像

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

      Lane 1 : F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates
      Lane 2 : F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates. Then the membrane was incubated with phosphatase.

      Lysates/proteins at 15 µg per lane.

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size : 48 kDa
      Observed band size : 50 kDa (why is the actual band size different from the predicted?)


      Exposure time : 1 minute

      Blocking and diluting buffer: 5% NFDM/TBST.

    • Ab52903 staining Smad3 in Human stomach tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraffin and antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, Ph9.0). Samples were incubated with primary antibody at a 1/200 dilution. A ready to use rabbit specific IHC polymer detection kit HRP/DAP (ab209101). Hematoxylin was used as a counterstain. Nuclear and weakly cytoplasmic staining on human stomach without alkaline phosphatase treatment (image A). No signal can be detected when tissues were treated with alkaline phosphatase (image B).

    • Ab52903 staining Smad3 in Mouse kidney tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraffin and antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1/200 dilution. A ready to use rabbit specific IHC polymer detection kit HRP/DAP (ab209101). Hematoxylin was used as a counterstain. Nuclear and weakly cytoplasmic staining on mouse kidney without alkaline phosphatase treatment (image A). No signal can be detected when tissues were treated with alkaline phosphatase (image B).

    • Immunocytochemistry/Immunofluorescence analysis of A549 +/- TGFβ (5ng/ml, 24h) and A549 + TGFβ (5ng/ml, 24h) + Lamda phosphatase (LP) cells. Smad3 (phospho S423 + S425) was labelled with ab52903 at a dilution of 1/100 dilution, while Smad3 was labelled with ab207447 at a dilution of 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton X-100. ab150077 (goat anti-rabbit IgG Alexa Fluor® 488) (1/1000) was used as the secondary antibody. The cells were co-stained with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) 1/200. Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

    • All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution

      Lane 1 : A549 whole cell lysate
      Lane 2 : A549 treated with 5ng/ml TGF-ß1 for 24 hours whole cell lysate
      Lane 3 : A549 treated with 5ng/ml TGF-ß1 for 24 hours whole cell lysate, the membrane was incubated with alkaline phosphatase

      Lysates/proteins at 10 µg per lane.

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size : 48 kDa
      Observed band size : 55 kDa (why is the actual band size different from the predicted?)

      Blocking and dilution buffer: 5% NFDM/TBST.

    • All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution

      Lane 1 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad3 non-phospho peptide
      Lane 2 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad3 (phospho S423/425) peptide
      Lane 3 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad2 non-phospho peptide
      Lane 4 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad2 (phospho S465/467) peptide
      Lane 5 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad1 non-phospho peptide
      Lane 6 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad1 (phospho S465/467) peptide

      Lysates/proteins at 10 µg per lane.

      Secondary
      Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/2000 dilution

      Predicted band size : 48 kDa
      Observed band size : 55 kDa (why is the actual band size different from the predicted?)


      Exposure time : 3 minutes

      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    • Dot blot analysis of Smad 3 (phospho S423 + S425) phospho peptide (Lane 1), Smad 3 (phospho S423) phospho peptide (Lane 2), Smad 3 (phospho S425) phospho peptide (Lane 3) and Smad 3 non-phospho peptide (Lane 4) labelling Smad 3 (phospho S423 + S425) with ab52903 at a dilution of 1/1000. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000. Blocking and dilution buffer: 5% NFDM /TBST.

    • Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.
    • 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

    • 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.
    • 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

    • 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

    参考文献

    This product has been referenced in:
    • Lai CM  et al. Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion. J Cell Biol 216:1439-1453 (2017). IHC ; Drosophila melanogaster . Read more (PubMed: 28363970) »
    • Han Y  et al. Mesenchymal Cell Reprogramming in Experimental MPLW515L Mouse Model of Myelofibrosis. PLoS One 12:e0166014 (2017). IF ; Mouse . Read more (PubMed: 28135282) »

    See all 97 Publications for this product

    レビューと Q&A

    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

    投稿 Jan 30 2014

    Application
    Western blot
    Sample
    Bos taurus Cell lysate - whole cell (Mammary epithelial cells BME-UV1 and MAC-T)
    Gel Running Conditions
    Reduced Denaturing (10)
    Loading amount
    50 µg
    Treatment
    5 ng/mL TGF-beta 1 from 0 to 48 hours
    Specification
    Mammary epithelial cells BME-UV1 and MAC-T
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Username

    Abcam user community

    Verified customer

    投稿 Jul 12 2017

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Mouse prostate gland)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Sodium Citrate 10mM pH=6.0
    Permeabilization
    Yes - PBS with 0.3% Triton
    Specification
    Mouse prostate gland
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
    Fixative
    Formaldehyde
    Username

    Abcam user community

    Verified customer

    投稿 Aug 11 2015

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Human prostate cancer cell)
    Gel Running Conditions
    Non-reduced Denaturing (10%)
    Loading amount
    30 µg
    Treatment
    TGF beta 5ng/ml
    Specification
    Human prostate cancer cell
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    投稿 Jul 27 2015

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Blocking step
    Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: citrate buffer, PH=6
    Sample
    Mouse Tissue sections (mouse)
    Specification
    mouse
    Permeabilization
    No
    Fixative
    Formaldehyde
    Username

    Abcam user community

    Verified customer

    投稿 Feb 17 2015

    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

    Dr. Francesco Elia Marino

    Verified customer

    投稿 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

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