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Products:Signal Transduction >> Signaling Pathway >> Nuclear Signaling >> SMADs
Overview
Product name
Anti-Smad3 antibody - ChIP Grade
See all Smad3 products (21) ...
Description
Rabbit polyclonal to Smad3 - ChIP Grade
Specificity
This antibody does not cross-react with other Smad proteins.
Tested applications
ChIP, IP, ICC/IF, WB, IHC-Pmore details
Cross reactivity
Reacts with
Mouse, Rat, Human
Immunogen
Synthetic peptide, corresponding to amino acids 192-211 of Human Smad3
Positive control
Normal breast or breast carcinoma.
Properties
Form
Liquid
Storage instructions
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage buffer
Preservative: 0.1% Sodium Azide
Constituents: 1% BSA, 10mM PBS, pH 7.4
Concentration
Concentration information loading...
Purity
Immunogen affinity purified
Purification notes
This antibody is affinity purified.
Clonality
Polyclonal
Isotype
IgG
Research areas
Cancer >> Cancer Metabolism >> Response to hypoxia
Epigenetics and Nuclear Signaling >> ChIP'ing antibodies >> ChIP'ing antibodies
Cancer >> Signal transduction >> Nuclear signaling >> SMAD family
Stem Cells >> Signaling Pathways >> TGF beta >> Cytoplasmic
Epigenetics and Nuclear Signaling >> Nuclear Signaling Pathways >> SMADs
Signal Transduction >> Signaling Pathway >> Nuclear Signaling >> SMADs
Applications
Show applications key
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: Use at an assay dependent dilution.
IP: Use at an assay dependent dilution.
ICC/IF: Use a concentration of 1 µg/ml
WB: Use at an assay dependent dilution. Predicted molecular weight: 48 kDa.
IHC-P: 1/100Perform 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.
Involvement in disease
Defects in SMAD3 may be a cause of colorectal cancer (CRC) [MIM:114500].
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.
Acetylation in the nucleus by EP300 in the MH2 domain regulates positively its transcriptional activity and is enhanced by TGF-beta.
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. 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.
Target information above from: UniProt accessionP84022
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010).
Alternative names
- DKFZP586N0721 antibody
- DKFZp686J10186 antibody
- hMAD 3 antibody
see all
Database links
- Entrez Gene: 4088 Human
- Entrez Gene: 17127 Mouse
- Entrez Gene: 25631 Rat
- Omim: 603109 Human
- SwissProt: P84022 Human
- SwissProt: Q8BUN5 Mouse
- SwissProt: P84025 Rat
- Unigene: 36915 Human
see all
Anti-Smad3 antibody - ChIP Grade images:
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Smad3 antibody (ab28379)
Ab28379, at a 1/100 dilution, staining Smad3 in formalin fixed paraffin embedded human breast carcinoma sections by Immunohistochemistry.
Western blot - Smad3 antibody (ab28379)
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
Goat polyclonal Secondary Antibody to 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
ChIP - Smad3 antibody - ChIP Grade (ab28379)
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.
This image is courtesy of an anonymous Abreview
Immunocytochemistry/ Immunofluorescence - Smad3 antibody - ChIP Grade (ab28379)
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.
Immunocytochemistry/ Immunofluorescence - Smad3 antibody - ChIP Grade (ab28379)
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.
This image is a courtesy of Aaron Gardner
References for Anti-Smad3 antibody - ChIP Grade (ab28379)
This product has been referenced in:
- van de Laar IMet al. Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis. Nat Genet 43:121-6 (2011). IHC-P; Human.Read more (PubMed: 21217753) »
- Gressner OAet al. Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein. World J Gastroenterol 17:151-63 (2011). WB; Rat.Read more (PubMed: 21245987) »
See all 19 publications for this product
Publishing research using ab28379? Please let us know so that we can cite the reference in this datasheet
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"