Product nameAnti-Smad3 antibody - ChIP Grade
See all Smad3 primary antibodies
DescriptionRabbit polyclonal to Smad3 - ChIP Grade
Tested applicationsSuitable for: ChIP, IP, ICC/IF, IHC-Fr, WB, IHC-Pmore details
Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide within Human Smad3 aa 150-250. The exact sequence is proprietary.
Database link: P84022
- Normal breast or breast carcinoma.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Storage bufferpH: 7.6
Preservative: 0.1% Sodium azide
Constituents: PBS, 1% BSA
Concentration information loading...
PurityImmunogen affinity purified
Purification notesThis antibody is affinity purified.
ChIP Related Products
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 concentration.|
|IP||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
|WB||Use at an assay dependent concentration. Predicted molecular weight: 48 kDa.|
|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.
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
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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
All lanes : 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?
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.
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.
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.
Ab28379, at a 1/100 dilution, staining Smad3 in formalin fixed paraffin embedded human breast carcinoma sections by Immunohistochemistry.
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.
This product has been referenced in:
- Chen T et al. PAK4 phosphorylates fumarase and blocks TGF-ß-induced cell growth arrest in lung cancer cells. Cancer Res N/A:N/A (2019). Read more (PubMed: 30683654) »
- Naciri I et al. Genetic screens reveal mechanisms for the transcriptional regulation of tissue-specific genes in normal cells and tumors. Nucleic Acids Res 47:3407-3421 (2019). Read more (PubMed: 30753595) »