Product nameAnti-Smad3 (phospho S213) antibody
See all Smad3 primary antibodies
DescriptionRabbit polyclonal to Smad3 (phospho S213)
SpecificityThis antibody detects endogenous levels of Smad3 only when phosphorylated at serine 213.
Tested applicationsSuitable for: ICC, WB, ELISA, ICC/IFmore details
Species reactivityReacts with: Human
Predicted to work with: Mouse, Rat
A synthesized phosphopeptide derived from human Smad3 around the phosphorylation site of serine 213 (PMSPPA)
- Extracts from HT29 cells This antibody gave a positive result in IF/ICC when used in the following formaldehyde fixed cell lines: A549.
Storage instructionsShipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.
Storage bufferpH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 50% Glycerol, 0.87% Sodium chloride
Without Mg2+ and Ca2+
Concentration information loading...
PurityImmunogen affinity purified
Purification notesThe antibody was affinity purified from rabbit antiserum by affinity chromatography using epitope specific phosphopeptide. The antibody against non phosphopeptide was removed by chromatography using non phosphopeptide corresponding to the phosphorylation site.
Our Abpromise guarantee covers the use of ab63403 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC||Use at an assay dependent concentration.|
|WB||1/500 - 1/1000. Detects a band of approximately 48 kDa (predicted molecular weight: 48 kDa).|
|ICC/IF||Use a concentration of 10 µg/ml.|
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.
Poly-ADP-ribosylated by PARP1 and PARP2. ADP-ribosylation negatively regulates SMAD3 transcriptional responses during the course of TGF-beta signaling.
Cellular localizationCytoplasm. 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
- DKFZP586N0721 antibody
- DKFZp686J10186 antibody
- hMAD 3 antibody
All lanes : Anti-Smad3 (phospho S213) antibody (ab63403) at 1/500 dilution
Lane 1 : Extracts from HT29 cells
Lane 2 : Extracts from HT29 cells plus phospho peptide
Predicted band size: 48 kDa
Observed band size: 48 kDa
The amount of positive control loading for the WB is 5-30 ug of total protein. The amount of the peptide is 5-10 ug.
ICC/IF image of ab63403 stained A549 cells. The cells were 4% formaldehyde 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 ab63403 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
ab63403 staining Smad3 (phospho S213) in Human A549 cells by Immunocytochemistry. Cells were fixed using the HOPE method and blocked for 5 minutes at 25°C. Samples were incubated with primary antibody (1/100) for 1 hour at 25°C. An undiluted HRP-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.
This product has been referenced in:
- Bai Y et al. Mesenchymal Stem Cells Reverse Diabetic Nephropathy Disease via Lipoxin A4 by Targeting Transforming Growth Factor ß (TGF-ß)/smad Pathway and Pro-Inflammatory Cytokines. Med Sci Monit 25:3069-3076 (2019). Read more (PubMed: 31023998) »
- Liu F et al. Bone morphogenetic protein and activin membrane-bound inhibitor suppress bone cancer progression in MG63 and SAOS cells via regulation of the TGF-ß-induced EMT signaling pathway. Oncol Lett 16:5113-5121 (2018). Read more (PubMed: 30250579) »