Overview

  • Product name
    Anti-Smad3 antibody - ChIP Grade
    See all Smad3 primary antibodies
  • Description
    Rabbit polyclonal to Smad3 - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ChIP, IP, ICC/IF, IHC-Fr, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Smad3 aa 150-250. The exact sequence is proprietary.
    Database link: P84022

  • Positive control
    • Normal breast or breast carcinoma.

Properties

Applications

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.

Application Abreviews Notes
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.

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
    modifications
    Phosphorylated 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
  • Alternative names
    • DKFZP586N0721 antibody
    • DKFZp686J10186 antibody
    • hMAD 3 antibody
    • hMAD-3 antibody
    • hSMAD3 antibody
    • HSPC193 antibody
    • HST17436 antibody
    • JV15 2 antibody
    • JV15-2 antibody
    • JV152 antibody
    • LDS1C antibody
    • LDS3 antibody
    • MAD (mothers against decapentaplegic Drosophila) homolog 3 antibody
    • MAD homolog 3 antibody
    • Mad homolog JV15 2 antibody
    • Mad protein homolog antibody
    • MAD, mothers against decapentaplegic homolog 3 antibody
    • Mad3 antibody
    • MADH 3 antibody
    • MADH3 antibody
    • MGC60396 antibody
    • Mothers against decapentaplegic homolog 3 antibody
    • Mothers against DPP homolog 3 antibody
    • SMA and MAD related protein 3 antibody
    • SMAD 3 antibody
    • SMAD antibody
    • SMAD family member 3 antibody
    • SMAD, mothers against DPP homolog 3 antibody
    • Smad3 antibody
    • SMAD3_HUMAN antibody
    see all

Images

  • 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

    Secondary
    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?



    Secondary antibody - anti-rabbit HRP (ab6721)

    See Abreview

  • 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.

    See Abreview

  • 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.

    See Abreview

  • 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.

    See Abreview

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human idiopathic pulmonary fibrosis lung sections, staining Smad3 with ab28379.

References

This product has been referenced in:
  • Zhang H  et al. Renal-protective effect of thalidomide in streptozotocin-induced diabetic rats through anti-inflammatory pathway. Drug Des Devel Ther 12:89-98 (2018). Read more (PubMed: 29386886) »
  • Zhang P  et al. Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription. Front Pharmacol 9:209 (2018). Read more (PubMed: 29593538) »
See all 98 Publications for this product

Customer reviews and Q&As

1-10 of 25 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (brain)
Specification
brain
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C

Dr. Amelia Sanchez-Capelo

Verified customer

Submitted Apr 24 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Tissue lysate - whole (Left Ventricle)
Loading amount
40 µg
Specification
Left Ventricle
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Dawn Bowles

Verified customer

Submitted Dec 28 2012

Answer

Thank you for your inquiry.

I am not aware that a primary antibody is used when doing a gel supershift (EMSA).

How are you planning to use the primary antibody?

We do not have antibodies tested and guaranteed for EMSA, but if you would like to use one, I assume that an antibody tested for IP would be the right choice.

If you would like to blot the EMSA gel as a control if the protein actually is bound to the DNA of interest, I suggest to choose an antibody that is tested for WB. Since the protein has to be in is native form to bind the DNA element, I recommend to choose an antibody that is also tested for IP or IHC, since then it is more likely that the antibody will bind the native protein.

Unfortunately, we do not test if an antibody binds to native protein for all our antibodies.

Please do not hesitate to contact me again with the requested information if you would like to have some more help choosing an antibody.

Read More

Question

First, the lab thanks you and Abcam for the lovely brownies!

Second, I have obtained A549 cells and HeLa cells for use in controls with RAW264.7 nuclear extracts. I have not done extensive studies to figure out the antibody problem. However, the data agree with the idea that RAW264.7 cells, control or treated, do not express Smad3. I have attached these results to this email if it is of interest to you and/or Abcam.

The large image file contains the contents of the wells/treatments/and antibodies used to detect the different Smad proteins and the phosphorylated Smad proteins. The second file, Abcam Smad3 signal, shows detection of a 55kD protein in A549 cytoplasmiclysates, +/-TGFb. This signal was not detected in HeLa cytoplasmic orRAW264.7 nuclearlysates, +/-TGFb. The Smad2and3 file, shows single Smad2 and Smad3 signals in the A549 lysates, but HeLa and RAW lysates show a single band. Combining the fact that the Smad3 primary amino acid sequence is shorter than that of Smad2 and I cannot detect Smad3 in Raw cells, I believe Raw cells only express Smad2. I attached the P-Smad2and3 file to show that TGFb treatment of all cell types results in the specific phosphorylation of Smad2 and not Smad3.

Since our lab does not use A549, I am not interested in increasing the signal observed in attached LICOR images. However, I think switchingblocking buffers, I used 5% milk/TBS +/-T,as you initially suggested could work for others. Again, thank you for the brownies, we will consider Abcam a reliable source of primary antibodies in the future.

Thanks again,

Read More
Answer

Thanks so much for getting back to me, and I apologize for the delay as I've been out of the office.
You're very welcome for the brownies! I know that this has been frustrating and you have spent a lot of effort and time working on this project. We are grateful for the the images and for the information about Smad3 expression. I will add this information to the datasheet in order to help other researchers.
If there is anything else that we can do for you now or in the future, please do let me know. I wish you the best of luck with your research!

Read More

Answer

DISCOUNT CODE FOR ab28379:**********
Expiration date: *************

I am very pleased to hear you would like to accept our offer and test ab28379 in flow cytometry. This code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for flow cytometry and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.


Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Read More

Answer

Thank you for your reply.

The only other phospho-smad3 (S423+S425) we have are:

ab11825 - https://www.abcam.com/Smad3-phospho-S423-S425-antibody-ab118825.html

ab52903 - https://www.abcam.com/Smad3-phospho-S423-S425-antibody-EP823Y-ab52903.html



Please let me know which one you would like to test and I will then send you the testing discount codes for it.

Also for the other antibodies, below are some selections:

PRKACA,

ab124390 - https://www.abcam.com/PRKACA-antibody-X1-ab124390.html

Stat3
ab119352 - https://www.abcam.com/STAT3-antibody-9D8-ab119352.html

phospho-Stat3
ab30647 - https://www.abcam.com/STAT3-phospho-S727-antibody-ab30647.html
ab30646 - https://www.abcam.com/STAT3-phospho-Y705-antibody-ab30646.html



Above are only a few of the antibodies that we have for these targets, so please feel free to search and see if you can find a more suitable alternative. Also non have been tested in flow cytometry and so would qualify for a testing discount code.

Please let me know if there is anything else I can help you with.

Read More

Answer

Thank you very much for contacting Abcam.

The isotype control that I would recommend for your flow cytometry experiments is:

https://www.abcam.com/Rabbit-IgG-ChIP-Grade-ab37415.html



Below is the infromation about the testing discount program, you would be eligbale to get a testing disount for all 3 antibodies:

https://www.abcam.com/TGF-beta-Receptor-I-antibody-ab125310.html
https://www.abcam.com/Smad3-phospho-S423-S425-antibody-ab51451.html
https://www.abcam.com/Smad3-antibody-ChIP-Grade-ab28379.html



To our knowledge, ab125310/ab28379/ab51451 has not been tested in Flow Cytometry. Therefore, I can offer a discount off a future purchase if you buy ab125310/ab28379/ab51451 now, test it in Flow Cytometry and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free PRIMARY ANTIBODY for each antibody that is tested in Flow Cytometry.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab125310/ab28379/ab51451 in Flow Cytometry. I will then send a discount code. This code must be issued before purchasing ab125310/ab28379/ab51451 so please wait for my reply before ordering.

2. Purchase ab125310/ab28379/ab51451 either by phone, fax, or online (www.abcam.com).

3. Test it in Flow Cytometry.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any each PRIMARY ANTIBODY ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab125310/ab28379/ab51451 turns out to be unsuitable for Flow Cytometry, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

Read More

Answer

Thanks for your reply.
I agree that running a known positive control is a good idea in this case, just in case the cells are expressing Smad 2 exclusively. Ab28379 has been tested in Western blot against Jurkat cell lysate, A549 lysate, and MDA231 cells, though if you don't have access to any of these cell lines I'll do some literature searching and see if there are others that might work.
Please let me know what you think, and keep me updated about any new results with this antibody. I look forward to hearing from you.

Read More

Question

I have attached my preliminary Smad3 western blot. I added one dual color image and one black/white image. If you have any questions regarding the blots, please let me know and I will address them asap.

I ran 30ugeach of untreated and TGFb treated (2h) nuclear lysates on 12% BioRad TGX gels. Again,isolated nuclei were lysed in SDS sample buffer without BPB,boiled for 5 min, and sonicated.Prior to loading I add SDS sample buffer with BPB and heat for 5 min at 70 degrees and spin before gel loading. I electrotransferred for 30 min at 100V to both nitrocellulose and PVDF-FL membranes. I also Syprostained (Invitrogen)the blots prior to blocking. This step does not interfere with LICOR imaging in our experience.

I blocked the blots for 1 hour at room temperature with shaking in 5% nonfat milk/ TBS. I incubated the blots overnight at 4 degrees, with shaking, in the indicatedSmad3 (ab28379) dilutions in 5% nonfat milk/TBST. I washed the blots in TBST at room temperature with shaking, 4X 5 min each wash. I then incubated the blots inTBP antibody diluted in5% nonfat milk/TBST, for 1 hour at room temperature. After washing as described previously, I incubated the blots for one hourin 5% nonfat milk/TBST, 0.01% SDS, and 1:20,000 each anti-rabbit 800 and anti-mouse 700.I washed the blots again, rinsed them in two changes of TBS and scanned. The signal intensity for 700 was 5 and was 8 for 800.

Again, I will gladly try using Odyssey Blocking Buffer and BSA for blocking/antibody dilution solutions. If you could send any recommendations along, they would be greatly appreciated.

Read More
Answer

Thanks for getting back to me and sending the protocol details and images.
I've gone through the protocol and I would recommend going ahead and trying the BSA and Odyssey buffers for blocking, as you've already tried a couple of different membranes and the rest of the protocol looks very good. I'm looking on the Licor website to see if there is anything else they would recommend in this case, but if none of the blots look good in any of the blocking buffers, I would be happy to send a new lot of the antibody or a different SMAD3 antibody to try.
I am sorry I don't have a great explanation for these results at this point, but I am hopeful that we can resolve this promptly. Please let me know if you have any questions or need anything else in the meantime.

Read More

Question
Answer

Thank you for your call today and for letting us know about the trouble with ab28379.
As we discussed, you can send the Western blot image in a reply to this email, and I will be happy to look into this problem.
I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that you need, and I'll be happy to help you further.

Read More

1-10 of 25 Abreviews or Q&A

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