Anti-Smad3 (phospho S423 + S425) [EP823Y] antibody (ab52903)
- Product nameAnti-Smad3 (phospho S423 + S425) [EP823Y] antibodySee all Smad3 primary antibodies ...
- DescriptionRabbit monoclonal [EP823Y] to Smad3 (phospho S423 + S425)
- Specificityab52903 detects Smad3 phosphorylated on Serine 423 and Serine 425. It may also detect Smad1, Smad2 and Smad5 phosphorylated at the equivalent sites.
- Tested applicationsWB, ICC/IF, ELISA, IHC-Fr, IHC-P more details
- Species reactivityReacts with: Mouse, Human
Does not react withRat
A synthetic phospho specific peptide corresponding to residues surrounding Ser423 and Ser425 of human Smad3.
- Positive control
- WB: HL-60 cell lysates IHC-P: Human liver carcinoma ICC/IF: HeLa cells
- General notesProduced under U.S. Patent No. 5,675,063.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%
- PurityTissue culture supernatant
- Clonality Monoclonal
- Clone numberEP823Y
Our Abpromise guarantee covers the use of ab52903 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||WB: 1/2000. Predicted molecular weight: 48 kDa.|
|ICC/IF||ICC/IF: 1/100 - 1/250.|
|ELISA||ELISA: Use at an assay dependent concentration.|
|IHC-Fr||IHC-Fr: Use at an assay dependent concentration. PubMed: 23667656|
|IHC-P||IHC-P: 1/100 - 1/250.|
- 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 (phospho S423 + S425) [EP823Y] antibody images
Predicted band size : 48 kDa
All lanes : Anti-Smad3 (phospho S423 + S425) [EP823Y] antibody (ab52903) at 1/2000 dilution
Lane 1 : (A) HL-60 cell lysates at 10µg untreated
Lane 2 : (B) HL-60 cell lysates at 10µg treated with TGF.
Predicted band size : 48 kDa
Observed band size : 55 kDa (why is the actual band size different from the predicted?)
Additional bands at : 45 kDa. We are unsure as to the identity of these extra bands.
Immunofluorescent staining of Smad3 in HeLa cells using ab52903 at 1/100 dilution.
Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.
All lanes : Anti-Smad3 (phospho S423 + S425) [EP823Y] antibody (ab52903) at 1/1000 dilution
Lane 1 : Lysate prepared from untreated human A549 cells
Lane 2 : Lysate prepared from untreated human A549 cells for 30min
Lane 3 : Lysate prepared from TGF-ß1 cells at 10ng/ml for 30min
Lane 4 : Lysate prepared from TNF-a cells at 20ng/ml for 30min
Lane 5 : Lysate prepared from TGF-ß1 and TNF-a cells at above doses for 30min
Lane 6 : Blank DMEM media
Lysates/proteins at 20 µg per lane.
Donkey polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab16284)
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 48 kDa
Observed band size : 48 kDa
Exposure time : 1 hour
This image is a courtesy of Aaron Gardner
ab52903 staining Smad3 (phospho S423 + S425) in human TII Pneumocyte A549c ells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton x100 before blocking with 3% BSA 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. A TRITC-conjugated goat polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution.
References for Anti-Smad3 (phospho S423 + S425) [EP823Y] antibody (ab52903)
This product has been referenced in:
- Meyer C et al. Caveolin-1 abrogates TGF-ß mediated hepatocyte apoptosis. Cell Death Dis 4:e466 (2013). WB ; Mouse . Read more (PubMed: 23328673) »
- 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) »