Overview

  • Product name

    Anti-Smad3 antibody - ChIP Grade
    See all Smad3 primary antibodies
  • Description

    Rabbit polyclonal to Smad3 - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ChIP, IP, ICC/IF, IHC-Fr, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Smad3 aa 150-250. The exact sequence is proprietary.
    Database link: P84022

  • Positive control

    • Normal breast or breast carcinoma.

Properties

Applications

Our Abpromise guarantee covers the use of ab28379 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
ChIP Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
IHC-Fr Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 48 kDa.
IHC-P 1/100. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

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

Images

  • All lanes : Anti-Smad3 antibody - ChIP Grade (ab28379) at 1/3000 dilution

    Lane 1 : HGL-5 cells: no additives
    Lane 2 : HGL-5 cells: + activin A
    Lane 3 : HGL-5 cells: + Cyclophilin, siRNA Control
    Lane 4 : HGL-5 cells: + activin A + Cyclophilin
    Lane 5 : HGL-5 cells: non targeting, negativ control for siRNA
    Lane 6 : HGL-5 cells: plus activin A, negative control for siRNA
    Lane 7 : HGL-5 cells: plus siRNA Smartpool
    Lane 8 : HGL-5 cells: + activin A +siRNA Smartpool
    Lane 9 : Jurkat cells

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/50000 dilution

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



    Secondary antibody - anti-rabbit HRP (ab6721)

    See Abreview

  • ab28379 staining Smad3 in Mouse brain tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 10% serum for 60 minutes at 22°C. Samples were incubated with primary antibody (1/100) for 12 hours at 4°C. An AlexaFluor®488-conjugated Mouse anti-rabbit polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab28379 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab28379, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Chromatin was prepared from the nuclear cell lysate of a human myofibroblast cell line according to the X-ChIP protocol; cross-linking with formaldehyde for 10 minutes. ab28379 was diluted 1/16 (RIPA 0.1% SDS) and incubated with the sample for 16 hours at 4°C. ab28379 IP was used as the positive control and rabbit IgG pool IP was used as the negative control. Smad3 expression was enhanced by 2ng/ml TGFbeta 2. The immunoprecipitated DNA was quantified by real time PCR.

    See Abreview

  • Ab28379, at a 1/100 dilution, staining Smad3 in formalin fixed paraffin embedded human breast carcinoma sections by Immunohistochemistry.
  • ab28379 staining Smad3 in human 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 was done 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. An TRITC-conjugated goat polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution.

    See Abreview

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human idiopathic pulmonary fibrosis lung sections, staining Smad3 with ab28379.

References

This product has been referenced in:

  • Chen T  et al. PAK4 phosphorylates fumarase and blocks TGF-ß-induced cell growth arrest in lung cancer cells. Cancer Res N/A:N/A (2019). Read more (PubMed: 30683654) »
  • Naciri I  et al. Genetic screens reveal mechanisms for the transcriptional regulation of tissue-specific genes in normal cells and tumors. Nucleic Acids Res 47:3407-3421 (2019). Read more (PubMed: 30753595) »
See all 119 Publications for this product

Customer reviews and Q&As

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1-10 of 13 Abreviews

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (heart)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EDTA
Permeabilization
No
Specification
heart
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jul 31 2019

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (brain)
Specification
brain
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C

Dr. Amelia Sanchez-Capelo

Verified customer

Submitted Apr 24 2013

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

Dawn Bowles

Verified customer

Submitted Dec 28 2012

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (A549)
Total protein in input
500 µg
Specification
A549
Immuno-precipitation step
Protein G

Dr. Aaron Gardner

Verified customer

Submitted Dec 06 2010

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (A549, Human TII Pneumocyte cell line)
Specification
A549, Human TII Pneumocyte cell line
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C

Dr. Aaron Gardner

Verified customer

Submitted Nov 13 2009

Application
Western blot
Sample
Human Cell lysate - whole cell (A549 Cells)
Loading amount
20 µg
Specification
A549 Cells
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris gel (Invitrogen - NP0322))
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Dr. Aaron Gardner

Verified customer

Submitted Oct 05 2009

Application
ChIP
Sample
Human Cell lysate - nuclear (Myofibroblast cell line)
Specification
Myofibroblast cell line
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Detection step
Real-time PCR
Positive control
ab28379 IP
Negative control
rabbit IgG pool IP

Abcam user community

Verified customer

Submitted Aug 20 2009

Application
Western blot
Sample
Human Cell lysate - whole cell (MDA 231 cell line)
Loading amount
25 µg
Specification
MDA 231 cell line
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted May 21 2009

Application
Western blot
Sample
Human Cell lysate - whole cell (Human Renal Proximal Tubular cells)
Loading amount
10 µg
Specification
Human Renal Proximal Tubular cells
Gel Running Conditions
Reduced Denaturing (7.5 %)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Dr. Robert Jenkins

Verified customer

Submitted May 21 2009

Application
Western blot
Sample
Mouse Cell lysate - whole cell (NIH 3T3)
Loading amount
25 µg
Specification
NIH 3T3
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted May 20 2009

1-10 of 13 Abreviews

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