Anti-Smad3 antibody [AF9F7] (ab75512)
- Product nameAnti-Smad3 antibody [AF9F7]See all Smad3 primary antibodies ...
- DescriptionMouse monoclonal [AF9F7] to Smad3
- Tested applicationsIP, Flow Cyt, Sandwich ELISA, WB, ELISA more details
- Species reactivityReacts with: Mouse, Rat, Human
Predicted to work with: Chicken, Ferret, Zebrafish
Recombinant full length Smad3 protein (Human) purified from E.coli (His-Smad3)
- Positive control
- Hela, IMR32, A431, Jurkat and PC12 cell lysates
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPreservative: 0.03% Sodium Azide
Constituents: 50% Glycerol, 0.01% BSA, HEPES, 0.15mM Sodium chloride
- Concentration information loading...
- PurityAmmonium Sulphate Precipitation
- Clonality Monoclonal
- Clone numberAF9F7
- Light chain typekappa
Our Abpromise guarantee covers the use of ab75512 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||IP: Use a concentration of 5 µg/ml.|
|Flow Cyt||Flow Cyt: 1/100.|
|Sandwich ELISA||sELISA: Use a concentration of 5 µg/ml. Can be paired for Sandwich ELISA with Rabbit monoclonal [EP568Y] to Smad3 (ab40854). For sandwich ELISA, use this antibody as Capture at 5 µg/ml with Rabbit monoclonal [EP568Y] to Smad3 (ab40854) as Detection.|
|WB||WB: 1/5000. Detects a band of approximately 56 kDa (predicted molecular weight: 48 kDa).|
|ELISA||ELISA: Use at an assay dependent dilution.|
- 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 [AF9F7] images
All lanes : Anti-Smad3 antibody [AF9F7] (ab75512) at 1/5000 dilution
Lane 1 : Hela cell lysate
Lane 2 : IMR32 cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate
Lane 5 : PC12 cell lysate
Predicted band size : 48 kDa
Observed band size : 56 kDa (why is the actual band size different from the predicted?)
Overlay histogram showing HCT116 cells stained with ab75512 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75512, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Smad3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Smad3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab75512.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 56kDa; Smad3
References for Anti-Smad3 antibody [AF9F7] (ab75512)
This product has been referenced in:
- Nolan-Stevaux O et al. Endoglin requirement for BMP9 signaling in endothelial cells reveals new mechanism of action for selective anti-endoglin antibodies. PLoS One 7:e50920 (2012). WB ; Rat . Read more (PubMed: 23300529) »