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

Overview

• Product nameAnti-Smad3 (phospho S423 + S425) antibody [EP823Y]See all Smad3 primary antibodies ...
• Description
  Rabbit monoclonal [EP823Y] to Smad3 (phospho S423 + S425)
• Specificityab52903 only detects Smad3 phosphorylated on Serine 423 and Serine 425.
• Tested applicationsWB, ICC/IF, ELISA, IHC-P more details
• Species reactivity
  Reacts with: Mouse, Human
  

  Does not react with

  Rat
• Immunogen

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

• Positive controlWB: HL-60 cell lysates IHC-P: Human liver carcinoma ICC/IF: HeLa cells
• General notesProduced under U.S. Patent No. 5,675,063.

Properties

• FormLiquid
• Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
• Storage bufferPBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%
• PurityTissue culture supernatant
• Clonality Monoclonal
• Clone numberEP823Y
• IsotypeIgG
• Research Areas
  • Metabolism
  • Types of disease
  • Cancer

  • Metabolism
  • Pathways and Processes
  • Metabolism processes
  • Hypoxia

  • Stem Cells
  • Signaling Pathways
  • TGF beta
  • Cytoplasmic

  • Cancer
  • Signal transduction
  • Nuclear signaling
  • SMAD family

  • Cancer
  • Cancer Metabolism
  • Response to hypoxia

  • Signal Transduction
  • Signaling Pathway
  • Nuclear Signaling
  • SMADs

  • Epigenetics and Nuclear Signaling
  • Nuclear Signaling Pathways
  • SMADs

Applications

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 (By similarity). 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 (CRC) [MIM:114500]: A complex disease characterized by malignant lesions arising from the inner wall of the large intestine (the colon) and the rectum. Genetic alterations are often associated with progression from premalignant lesion (adenoma) to invasive adenocarcinoma. Risk factors for cancer of the colon and rectum include colon polyps, long-standing ulcerative colitis, and genetic family history. Note=The disease may be caused by mutations affecting the gene represented in this entry.
  Loeys-Dietz syndrome 3 (LDS3) [MIM:613795]: An aortic aneurysm syndrome with widespread systemic involvement. The disorder is characterized by the triad of arterial tortuosity and aneurysms, hypertelorism, and bifid uvula or cleft palate. Patients with LDS3 also manifest early-onset osteoarthritis. They lack craniosynostosis and mental retardation. Note=The disease is caused by mutations affecting the gene represented in this entry. SMAD3 mutations have been reported to be also associated with thoracic aortic aneurysms and dissection (TAAD) (PubMed:21778426). This phenotype is distinguised from LDS3 by having aneurysms restricted to thoracic aorta. As individuals carrying these mutations also exhibit aneurysms of other arteries, including abdominal aorta, iliac, and/or intracranial arteries (PubMed:21778426), they have been classified as LDS3 by the OMIM resource.
• 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
  modificationsPhosphorylated 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
  • Entrez Gene: 4088 Human
  • Entrez Gene: 17127 Mouse
  • Omim: 603109 Human
  • SwissProt: P84022 Human
  • SwissProt: Q8BUN5 Mouse
  • Unigene: 727986 Human
  • Unigene: 7320 Mouse

• Alternative names
  DKFZP586N0721 antibodyDKFZp686J10186 antibodyhMAD 3 antibody

  hMAD-3 antibodyhSMAD3 antibodyHSPC193 antibodyHST17436 antibodyJV15 2 antibodyJV15-2 antibodyJV152 antibodyLDS1C antibodyLDS3 antibodyMAD (mothers against decapentaplegic Drosophila) homolog 3 antibodyMAD homolog 3 antibodyMad homolog JV15 2 antibodyMad protein homolog antibodyMAD, mothers against decapentaplegic homolog 3 antibodyMAD3 antibodyMADH 3 antibodyMADH3 antibodyMGC60396 antibodyMothers against decapentaplegic homolog 3 antibodyMothers against DPP homolog 3 antibodySMA and MAD related protein 3 antibodySMAD 3 antibodySMAD antibodySMAD family member 3 antibodySMAD, mothers against DPP homolog 3 antibodySMAD3 antibodySMAD3_HUMAN antibody

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

• 

  Western blot - Smad3 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.
• 

  Immunocytochemistry/ Immunofluorescence - Smad3 antibody [EP823Y] (ab52903)
  Immunofluorescent staining of Smad3 in HeLa cells using ab52903 at 1/100 dilution.
• 

  Immunohistochemistry - Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.
• 

  Western blot - Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)
  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 polyclonal Secondary Antibody to 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

• 

  Immunocytochemistry/ Immunofluorescence - 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 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