Anti-Smad3 antibody - ChIP Grade (ab28379)
- Product nameAnti-Smad3 antibody - ChIP GradeSee all Smad3 primary antibodies ...
- DescriptionRabbit polyclonal to Smad3 - ChIP Grade
- SpecificityThis antibody does not cross-react with other Smad proteins.
- Tested applicationsChIP, IP, ICC/IF, IHC-Fr, WB, IHC-P more details
- Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide, corresponding to amino acids 192-211 of Human Smad3
- Positive control
- Normal breast or breast carcinoma.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPreservative: 0.1% Sodium Azide
Constituents: 1% BSA, 10mM PBS, pH 7.4
- Concentration information loading...
- PurityImmunogen affinity purified
- Purification notesThis antibody is affinity purified.
- Clonality Polyclonal
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.
|ChIP||ChIP: Use at an assay dependent concentration.|
|IP||IP: Use at an assay dependent concentration.|
|ICC/IF||ICC/IF: Use a concentration of 1 µg/ml.|
|IHC-Fr||IHC-Fr: Use at an assay dependent concentration.|
|WB||WB: Use at an assay dependent concentration. Predicted molecular weight: 48 kDa.|
|IHC-P||IHC-P: 1/100. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.|
- 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. 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
Loeys-Dietz syndrome 3
- 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.
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.
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Anti-Smad3 antibody - ChIP Grade images
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.
Ab28379, at a 1/100 dilution, staining Smad3 in formalin fixed paraffin embedded human breast carcinoma sections by Immunohistochemistry.
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
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?)
This image is courtesy of an Abreview submitted by Mrs Birte Jablonski
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.
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.
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.
Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human idiopathic pulmonary fibrosis lung sections, staining Smad3 with ab28379.
References for Anti-Smad3 antibody - ChIP Grade (ab28379)
This product has been referenced in:
- Estarás C et al. RNA polymerase II progression through H3K27me3-enriched gene bodies requires JMJD3 histone demethylase. Mol Biol Cell 24:351-60 (2013). WB, ChIP, ICC/IF ; Mouse . Read more (PubMed: 23243002) »
- Chu IM et al. Expression of GATA3 in MDA-MB-231 triple-negative breast cancer cells induces a growth inhibitory response to TGFß. PLoS One 8:e61125 (2013). WB . Read more (PubMed: 23577196) »