• Product name

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

    Rabbit polyclonal to Smad3 - ChIP Grade
  • Host species

  • 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.



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.


  • 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

    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


  • 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

    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.


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

11-20 of 26 Abreviews or Q&A


Thank you for your call today and for letting us know about the trouble with ab28379.
As we discussed, you can send the Western blot image in a reply to this email, and I will be happy to look into this problem.
I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that you need, and I'll be happy to help you further.

Read More


Thank you for your email. Dr. Tanya is away this week so I am dealing with her inquiries.

I have read the case and would like to suggest the following;

- The image attached is a cut out image, it doesn't show the multiple bands, to me the band shown is the specific one, I would not say antibody is binding non-specifically. Try the antibody with following suggestions; the results will be much clear

- use antibody at 1/2000 dilution
- Try lysates 20 ug
- Block the membrane in 5% BSA (TBST)
- Wash the membrane 4 times, first wash 10 minutes and other three 5 minutes.
- Try the no primary control also; it will help to determine if secondary is binding non specifically.

I sure following these steps the image will be much nicer.

Read More


LOT NUMBER gr47182-2 ORDER NUMBER 961552 DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE •Species: Human •What’s cell line or tissue: Cell line •Cell extract or Nuclear extract: Cell extract •Purified protein or Recombinant protein: PRIMARY ANTIBODY •At what dilution(s) have you tested this antibody: 1:1000 •What dilution buffer was used: TBS-T •Incubation time: overnight •Incubation temperature: 4℃ •What washing steps were done: TBS-T wash 3*10min DETECTION METHOD ECl POSITIVE AND NEGATIVE CONTROLS USED NO ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION •What lysis buffer was used: 50mM Hepes PH 7.4 100mM NaCl 2mM Na3VO4 1mM EDTA 1% Triton •What protease inhibitors were used: cock tail SI P8340 •What loading buffer was used: •Phosphatase inhibitors •Did you heat the samples: temperature and time: 100 ℃, 10 min AMOUNT OF PROTEIN LOADED 15 μg ELECTROPHORESIS/GEL CONDITIONS •Gel percentage : 12% TRANSFER AND BLOCKING CONDITIONS •Buffer:TBS-T •Blocking agent: milk, BSA, serum, what percentage: 5% milk •Incubation time:1 hr •Incubation temperature:RT SECONDARY ANTIBODY •At what dilution(s) have you tested this antibody:1:5000 •Incubation time: 1 hr •Wash steps: TBS-T wash 3*10min •Fluorochrome or enzyme conjugate: •Do you know whether the problems you are experiencing come from the secondary? NO HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Concentration of primary antibody

Read More

Thank you for your enquiry regarding ab28379 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Cell type: Could you please clarify the cell type used for this study? Loading control: It would be great if the loading control lanes can be also provided. Cell viability: Has the cell viability been checked in each treatment group to test and make sure that the cells were still alive after the treatment with the Smad3 inducer? Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.  

Read More
Human Cell lysate - whole cell (A549)
Total protein in input
500 µg
Immuno-precipitation step
Protein G

Dr. Aaron Gardner

Verified customer

Submitted Dec 06 2010

Immunocytochemistry/ Immunofluorescence
Human Cell (A549, Human TII Pneumocyte cell line)
A549, Human TII Pneumocyte cell line
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

Western blot
Human Cell lysate - whole cell (A549 Cells)
Loading amount
20 µg
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

Human Cell lysate - nuclear (Myofibroblast cell line)
Myofibroblast cell line
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

Western blot
Human Cell lysate - whole cell (MDA 231 cell line)
Loading amount
25 µg
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

Western blot
Human Cell lysate - whole cell (Human Renal Proximal Tubular cells)
Loading amount
10 µg
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

Western blot
Mouse Cell lysate - whole cell (NIH 3T3)
Loading amount
25 µg
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

11-20 of 26 Abreviews or Q&A

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