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RecombinantRabMAb

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

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Reviews (17)Q&A (4)References (295)

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Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Dot Blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  • Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP823Y] to Smad3 (phospho S423 + S425)
  • Suitable for: WB, ICC/IF, IHC-P, Dot blot
  • Reacts with: Mouse, Human

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Overview

  • Product name

    Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]
    See all Smad3 primary antibodies
  • Description

    Rabbit monoclonal [EP823Y] to Smad3 (phospho S423 + S425)
  • Host species

    Rabbit
  • Specificity

    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.
  • Tested Applications & Species

    Application Species
    Dot
    Human
    ICC/IF
    Human
    IHC-P
    Mouse
    Human
    WB
    Mouse
    Human
    See all applications and species data
  • Immunogen

    corresponding to Human Smad3.

  • Positive control

    • WB: HL-60 treated with TGF-ß cell lysates; A549 untreated and treated with 5ng/ml TGF-ß1 for 24 hours whole cell lysates; F9 whole cell lysate. IHC-P: Human stomach and liver carcinoma tissue; Mouse kidney tissue; Environmental enteropathy (EE) duodenal biopsy. ICC/IF: TGFß treated A549 cells; PML+/+ mouse embryonic fibroblasts (MEFs) were transfected with either CTL-siRNAs or NDRG1-siRNAs; Mouse primary embryonic epicardial cells.
  • General notes

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

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP823Y
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • SMADs
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • SMADs
    • Stem Cells
    • Signaling Pathways
    • TGF beta
    • Cytoplasmic
    • Cancer
    • Signal transduction
    • Nuclear signaling
    • SMAD family
    • Cancer
    • Cancer Metabolism
    • Response to hypoxia
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia
    • Metabolism
    • Types of disease
    • Cancer

Associated products

  • Alternative Versions

    • Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] - BSA and Azide free (ab172202)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • HL-60 whole cell lysate (ab7914)
  • Recombinant Protein

    • Recombinant Human Smad3 protein (ab89353)
  • Related Products

    • Prestained Protein Ladder - Broad molecular weight (10-245 kDa) (ab116028)

Applications

The Abpromise guarantee

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.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Dot
Human
ICC/IF
Human
IHC-P
Mouse
Human
WB
Mouse
Human
All applications
Drosophila melanogaster
Application Abreviews Notes
WB (11)
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 (2)
1/100 - 1/250.
IHC-P (3)
1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

Dot blot
1/1000.
Notes
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.
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

Dot blot
1/1000.
  • Application notes
    Is unsuitable for Flow Cyt or 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).
    • 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

      • Entrez Gene: 4088 Human
      • Entrez Gene: 17127 Mouse
      • Omim: 603109 Human
      • SwissProt: P84022 Human
      • SwissProt: Q8BUN5 Mouse
      • Unigene: 727986 Human
      • Unigene: 742270 Human
      • Unigene: 7320 Mouse
      • 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

      Images

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

        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
        All lanes : 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.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

        Purified 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).

      • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

        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 purified 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.

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

        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
        All lanes : 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.

      • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        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.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

        Purified 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).

      • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        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 S463/465) peptide

        Lysates/proteins at 10 µg per lane.

        Secondary
        All lanes : 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.

      • Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Western blot - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)This image is a courtesy of Aaron Gardner
        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
        All lanes : 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

        See Abreview

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)Image from Syed S et al., PLoS Negl Trop Dis. 2018;12(2):e0006224. Fig 4.; doi: 10.1371/journal.pntd.0006224. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

        Representative IHC photomicrographs from an Environmental enteropathy (EE) duodenal biopsy showing p-SMAD3 staining (ab52903) in only the epithelium (arrows).

      • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)This image is courtesy of an anonymous Abreview

        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

      • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)Image from Tang MK et al., PLoS One. 2013;8(3):e59477. Fig 12.; doi: 10.1371/journal.pone.0059477. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

        TGF-β1 signaling is impaired in NDRG1-silenced MEFs. PML+/+ mouse embryonic fibroblasts (MEFs) were transfected with either CTL-siRNAs (A & B) or NDRG1-siRNAs (C & D) and induced with100 ng/ml TGF-β1. Immunofluorescent staining revealed intense nuclear staining for phosphorylated SMAD3 (SMAD3-P, ab52903) in CTL-siRNA treated MEFs (B) while only weak nuclear staining for MEFs treated with NDRG1-siRNA (D).

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.
      • Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
        Immunocytochemistry/ Immunofluorescence - Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)This image is a courtesy of Aaron Gardner

        ab52903 staining Smad3 (phospho S423 + S425) in human TII Pneumocyte A549 cells 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

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

        Dot blot analysis of human 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.

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

      Protocols

      To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

      Click here to view the general protocols

      Datasheets and documents

      • Datasheet
      • SDS
    • References (295)

      Publishing research using ab52903? Please let us know so that we can cite the reference in this datasheet.

      ab52903 has been referenced in 295 publications.

      • Qu S  et al. MicroRNA-194 reduces inflammatory response and human dermal microvascular endothelial cells permeability through suppression of TGF-ß/SMAD pathway by inhibiting THBS1 in chronic idiopathic urticaria. J Cell Biochem 121:111-124 (2020). PubMed: 31190349
      • Saric N  et al. The AHR pathway represses TGFß-SMAD3 signalling and has a potent tumour suppressive role in SHH medulloblastoma. Sci Rep 10:148 (2020). PubMed: 31924815
      • Zhou P  et al. Transforming growth factor beta (TGF-ß) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure. Int J Biol Sci 16:204-215 (2020). PubMed: 31929749
      • Nath SR  et al. MEF2 impairment underlies skeletal muscle atrophy in polyglutamine disease. Acta Neuropathol 140:63-80 (2020). PubMed: 32306066
      • Xiao Z  et al. RUNX3 inhibits the invasion and migration of esophageal squamous cell carcinoma by reversing the epithelial-mesenchymal transition through TGF-ß/Smad signaling. Oncol Rep 43:1289-1299 (2020). PubMed: 32323849
      View all Publications for this product

      Customer reviews and Q&As

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      1-10 of 21 Abreviews or Q&A

      Immunocytochemistry/ Immunofluorescence abreview for Anti-Smad3 (phospho S423 + S425) [EP823Y] antibody

      Excellent
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Jan 30 2014

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Excellent
      Abreviews
      Abreviews
      abreview image
      Application
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
      Sample
      Rat Tissue sections (Pulmonary alveolar type 2 cells)
      Antigen retrieval step
      Heat mediated - Buffer/Enzyme Used: pH9, 30min
      Permeabilization
      No
      Specification
      Pulmonary alveolar type 2 cells
      Blocking step
      Serum as blocking agent for 10 minute(s) · Concentration: 1.5% · Temperature: 24°C
      Fixative
      Formaldehyde
      Read More

      Abcam user community

      Verified customer

      Submitted Nov 09 2020

      Western blot abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Excellent
      Abreviews
      Abreviews
      abreview image
      Application
      Western blot
      Sample
      Zebrafish Tissue lysate - whole (Liver)
      Gel Running Conditions
      Non-reduced Denaturing (12% SDS-PAGE)
      Loading amount
      25 µg
      Treatment
      60 µM Doxycycline for 7 day
      Specification
      Liver
      Blocking step
      Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
      Read More

      Abcam user community

      Verified customer

      Submitted Apr 14 2020

      Western blot abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Good
      Abreviews
      Abreviews
      abreview image
      Application
      Western blot
      Sample
      Human Cell lysate - whole cell (melanoma)
      Gel Running Conditions
      Reduced Denaturing (10%)
      Loading amount
      20 µg
      Treatment
      TFG-beta1 for 1hr
      Specification
      melanoma
      Blocking step
      BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
      Read More

      Abcam user community

      Verified customer

      Submitted Jul 23 2019

      Western blot abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Excellent
      Abreviews
      Abreviews
      abreview image
      Application
      Western blot
      Sample
      Human Cell lysate - whole cell (vascular smooth muscle cells)
      Gel Running Conditions
      Reduced Denaturing (4-12%)
      Loading amount
      20 µg
      Treatment
      TGFb2 for 30 minutes
      Specification
      vascular smooth muscle cells
      Blocking step
      BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
      Read More

      Michele Cavalera

      Verified customer

      Submitted May 11 2018

      Western blot abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Good
      Abreviews
      Abreviews
      abreview image
      Application
      Western blot
      Sample
      Rat Tissue lysate - whole (Muscle)
      Gel Running Conditions
      Reduced Denaturing (9)
      Loading amount
      50 µg
      Specification
      Muscle
      Blocking step
      BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
      Read More

      Mr. Baptiste Jude

      Verified customer

      Submitted Feb 26 2018

      Western blot abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Excellent
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Jul 12 2017

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Excellent
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Aug 11 2015

      Western blot abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Good
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Jul 27 2015

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Smad3 (phospho S423 + S425) antibody [EP823Y]

      Excellent
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Feb 17 2015

      1-10 of 21 Abreviews or Q&A

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