Recombinant Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)
![Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-410719-anti-smad3-antibody-epr19686-chip-grade-western-blot-a549-wt-a549-smad3-knockout-hela-human-kidney.jpg "Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR19686] to Smad3 - ChIP Grade
- Suitable for: ChIP, WB, IP
- Knockout validated
- Reacts with: Mouse, Rat, Human, Recombinant fragment
Related conjugates and formulations
Overview
-
Product name
Anti-Smad3 antibody [EPR19686] - ChIP Grade
See all Smad3 primary antibodies -
Description
Rabbit monoclonal [EPR19686] to Smad3 - ChIP Grade -
Host species
Rabbit -
Tested applications
Suitable for: ChIP, WB, IPmore details -
Species reactivity
Reacts with: Mouse, Rat, Human, Recombinant fragment -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
-
Positive control
- WB: Human Smad3 recombinant protein; A549, HeLa, Jurkat, HEK-293, K562, BxPC-3, C6, PC-12 and NIH/3T3 whole cell lysates; human fetal kidney lysate; rat spleen and kidney lysates; mouse spleen lysate. IP: HeLa whole cell lysate. ChIP: Chromatin was prepared from HaCaT cells.
-
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
-
Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading... -
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR19686 -
Isotype
IgG -
Research areas
Associated products
-
Alternative Versions
-
Compatible Secondaries
-
Isotype control
-
KO cell lines
-
KO cell lysates
-
Recombinant Protein
-
Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab208182 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| ChIP |
Use 2 µg for 25 µg of chromatin.
|
|
| WB |
1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa).
|
|
| IP |
1/30.
|
| Notes |
|---|
|
ChIP
Use 2 µg for 25 µg of chromatin. |
|
WB
1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa). |
|
IP
1/30. |
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. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator. -
Involvement in disease
Colorectal cancer
Loeys-Dietz syndrome 3 -
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
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 localization
Cytoplasm. 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
-
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: 727986 Human
see all -
Alternative names
- DKFZP586N0721 antibody
- DKFZp686J10186 antibody
- hMAD 3 antibody
see all
Images
-
Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : SMAD3 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Human Kidney cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 48 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab208182 observed at 50 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab208182 was shown to react with Smad3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab208182 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
![ChIP - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-6-AntiSmad3-antibody-EPR19686--ChIP-GradeChIP.jpg)
ChIP - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)Chromatin was prepared from HaCaT (Human keratinocyte cell line) cells treated with 7ng/ml TGF-β for 1h and non-treated according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab208182 (red), and 20µl of protein A/G beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
The ChIP condition performed here is similar to the literature (PMID: 18245174).
-
![Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-384905-anti-smad3-antibody-epr19686-chip-grade-western-blot-human-lysate.jpg)
Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : SMAD3 knockout A549 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : SMAD3 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 48 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab208182 observed at 55 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab208182 was shown to react with Smad3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255431 (knockout cell lysate ab263834) was used. Wild-type and Smad3 knockout samples were subjected to SDS-PAGE. ab208182 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
![Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-263470-anti-smad3-antibody-epr19686-chip-grade-western-blot.jpg)
Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution
Lane 1 : Human Smad3 recombinant protein
Lane 2 : Human Smad2 active protein
Lysates/proteins at 0.01 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 48 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?
Exposure time: 1 secondBlocking/Dilution buffer: 5% NFDM/TBST.
Human Smad3 recombinant protein contains aa2-227 with GST and His-Tag®. This protein was made-in house.
-
![Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-263469-anti-smad3-antibody-epr19686-chip-grade-western-blot.jpg)
Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 5 : BxPC-3 (Human pancreas adenocarcinoma cell line) whole cell lysate
Lane 6 : Human fetal kidney lysate
Lysates/proteins at 10 µg per lane.
Secondary
Lanes 1-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 6 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution
Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1/6: 3 minutes; Lane 2-5: 15 seconds.
-
![Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-263468-anti-smad3-antibody-epr19686-chip-grade-western-blot.jpg)
Western blot - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)All lanes : Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182) at 1/1000 dilution
Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : Rat spleen lysate
Lane 5 : Rat kidney lysate
Lane 6 : Mouse spleen lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1/2: 30 seconds; Lane 3-6: 3 minutes.
-
![Immunoprecipitation - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-263467-anti-smad3-antibody-epr19686-chip-grade-immunoprecipitation.jpg)
Immunoprecipitation - Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)Smad3 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208182 at 1/30 dilution.
Western blot was performed from the immunoprecipitate using ab208182 at 1/1000 dilution.
VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate 10µg (Input).
Lane 2: ab208182 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208182 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.
-
![Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)](https://www.abcam.com/ps/products/208/ab208182/Images/ab208182-10-benefits-of-recombinant-antibodies.png)
Anti-Smad3 antibody [EPR19686] - ChIP Grade (ab208182)
Protocols
Datasheets and documents
-
SDS download
-
Datasheet download
Certificate of Compliance
References (6)
ab208182 has been referenced in 6 publications.
- Niu B et al. Interplay between transforming growth factor-ß and Nur77 in dual regulations of inhibitor of differentiation 1 for colonic tumorigenesis. Nat Commun 12:2809 (2021). PubMed: 33990575
- Li H et al. Aggravation of hepatic ischemia-reperfusion injury with increased inflammatory cell infiltration is associated with the TGF-ß/Smad3 signaling pathway. Mol Med Rep 24:N/A (2021). PubMed: 34132369
- Zhang Z et al. The Jumonji Domain-Containing Histone Demethylase Homolog 1D/lysine Demethylase 7A (JHDM1D/KDM7A) Is an Epigenetic Activator of RHOJ Transcription in Breast Cancer Cells. Front Cell Dev Biol 9:664375 (2021). PubMed: 34249916
- Wang L et al. miR-135a Suppresses Granulosa Cell Growth by Targeting Tgfbr1 and Ccnd2 during Folliculogenesis in Mice. Cells 10:N/A (2021). PubMed: 34440873
- Jiang C et al. Xanthohumol Inhibits TGF-ß1-Induced Cardiac Fibroblasts Activation via Mediating PTEN/Akt/mTOR Signaling Pathway. Drug Des Devel Ther 14:5431-5439 (2020). PubMed: 33324040
- Gong Y et al. Megakaryocyte-derived excessive transforming growth factor ß1 inhibits proliferation of normal hematopoietic stem cells in acute myeloid leukemia. Exp Hematol 60:40-46.e2 (2018). ChIP ; Mouse . PubMed: 29307605