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Phospho SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903 is a rabbit monoclonal antibody that is used in Smad3 + SMAD5 + SMAD2 western blotting, IHC and immunofluorescence. Suitable for human and mouse samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone EP823Y has been tried and trusted by researchers since 2007 and is cited in >660 publications
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
New 20 ul size available


Images

Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (AB52903), expandable thumbnail
  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (AB52903), expandable thumbnail
  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (AB52903), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (AB52903), expandable thumbnail
  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (AB52903), expandable thumbnail

Publications

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IPFlow CytDotWBICC/IFIHC-PChIC/CUT&RUN-seq
Human
Not recommended
Not recommended
Tested
Tested
Tested
Tested
Tested
Mouse
Not recommended
Not recommended
Expected
Tested
Expected
Tested
Expected

Not recommended
Not recommended

Species

Mouse, Human

Dilution info

-

Notes

-

Not recommended
Not recommended

Species

Mouse, Human

Dilution info

-

Notes

-

Tested
Tested

Species

Human

Dilution info

1/1000

Notes

-

Expected
Expected

Species

Mouse

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/2000

Notes

-

Species

Human

Dilution info

1/2000

Notes

-

Tested
Tested

Species

Human

Dilution info

1/100 - 1/250

Notes

-

Expected
Expected

Species

Mouse

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/100 - 1/250

Notes

The secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Species

Human

Dilution info

1/100 - 1/250

Notes

The secondary antibody is rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species

Human

Dilution info

-

Notes

-

Expected
Expected

Species

Mouse

Dilution info

Use at an assay dependent concentration.

Notes

-

Associated Products

Select an associated product type

15 products for Alternative Product

Target data

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.

Targets

SMAD5 phospho S463 + S465, SMAD2 phospho S465 + S467, SMAD1 phospho S463 + S465

Alternative names

Recommended products

Phospho SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903 is a rabbit monoclonal antibody that is used in Smad3 + SMAD5 + SMAD2 western blotting, IHC and immunofluorescence. Suitable for human and mouse samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone EP823Y has been tried and trusted by researchers since 2007 and is cited in >660 publications
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
New 20 ul size available

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EP823Y

Purification technique

Affinity purification Protein A

Specificity

This antibody detects Smad3 phosphorylated on Serine 423 and Serine 425. This Smad3 antibody may also detect Smad1, Smad2 and Smad5 phosphorylated at the equivalent sites.

Concentration
Loading...

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

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 batch production

For more information, read more on recombinant antibodies.

Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Smad3 also known as Mothers against decapentaplegic homolog 3 is a protein involved in the TGF-? signaling pathway. This protein weighs approximately 48 kilodaltons. It is mainly expressed in various tissues including heart lung kidney and skin. Smad3 mediates signals from the TGF-? receptors to regulate gene expression in the nucleus. This function is often studied using markers like p-Smad3 phospho-Smad3 and phospho-Smad which identify the phosphorylated forms important for its activation.

Biological function summary

Smad3 plays an important role in regulating cell proliferation differentiation and apoptosis. It is a part of the Smad complex that includes Smad2 and Smad4 which translocate to the nucleus to regulate the transcription of target genes. This activity is important for maintaining cellular functions and responding to external signals. Phosphorylation of Smad3 is an important step in its activation often studied using phospho-specific antibodies some of which are conjugated with Alexa Fluor 594 or Alexa Fluor 555 to facilitate visualization in research.

Pathways

Smad3 is an essential component of the TGF-? signaling pathway and also participates in the BMP pathway. In the TGF-? pathway it collaborates with related proteins such as Smad2 and Smad4. These proteins assemble upon receptor activation propagate the signal to the nucleus and regulate gene transcription. Smad3's function in both the TGF-? and BMP pathways demonstrates its role in controlling diverse cellular processes.

Associated diseases and disorders

Smad3 has been linked to fibrosis and cancer. In fibrosis the overactivation of Smad3 leads to excessive extracellular matrix production contributing to tissue scarring. Cancer research highlights its role in tumorigenesis where alterations in Smad3 expression or activity affect disease progression. The connection of Smad3 with other proteins like TGF-? receptors and Smad4 in these disorders emphasizes its importance in pathological processes.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

18 product images

  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail
    Image from Tang MK et al. PLoS One. 2013;8(3):e59477. Fig 12.; doi: 10.1371/journal.pone.0059477.

    Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Phospho Smad3 immunofluorescence staining of mouse embryonic fibroblasts using rabbit anti-Phospho Smad3 antibody

    TGF-β1 signaling is impaired in NDRG1-silenced MEFs. PML+/+ mouse embryonic fibroblasts (MEFs) were transfected with either CTL-siRNAs (A & B) or NDRG1-siRNAs (C & D) and induced with100 ng/ml TGF-β1. Immunofluorescent staining revealed intense nuclear staining for phosphorylated SMAD3 (SMAD3-P ab52903) in CTL-siRNA treated MEFs (B) while only weak nuclear staining for MEFs treated with NDRG1-siRNA (D).

  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail
    This image is courtesy of an anonymous customer review.

    Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Phospho Smad3 immunofluorescence staining of primary embyronic epicardial cells using rabbit anti-Phospho Smad3 antibody

    ab52903 staining Smad3 in mouse primary embryonic epicardial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% formaldehyde permeabilized with 0.5% Triton X-100 and blocked with PBS + 1% BSA + 10% goat serum + 0.1% Triton X-100 for 1 hour at 20°C. Samples were incubated with primary antibody (1/100 dilution in PBS + 1% BSA + 10% goat serum + 0.1% Triton X-100) for 16 hours at 4°C. An Alexa Fluor®488-conjugated goat anti-rabbit IgG polyclonal (1/200 dilution) was used as the secondary antibody.

  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Blocking and diluting buffer: 5% NFDM/TBST.

    All lanes: Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903) at 1/2000 dilution

    Lane 1: F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates at 15 µg

    Lane 2: F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates. Then the membrane was incubated with phosphatase. at 15 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 48 kDa

    Observed band size: 50 kDa

    Exposure time: 1min

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail
    Image from Syed S et al. PLoS Negl Trop Dis. 2018;12(2):e0006224. Fig 4.; doi: 10.1371/journal.pntd.0006224.

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Representative IHC photomicrographs from an Environmental enteropathy (EE) duodenal biopsy showing p-SMAD3 staining (ab52903) in only the epithelium (arrows).

  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Blocking and dilution buffer: 5% NFDM/TBST.

    All lanes: Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903) at 1/1000 dilution

    Lane 1: A549 whole cell lysate at 10 µg

    Lane 2: A549 treated with 5ng/ml TGF-β1 for 24 hours whole cell lysate at 10 µg

    Lane 3: A549 treated with 5ng/ml TGF-β1 for 24 hours whole cell lysate, the membrane was incubated with alkaline phosphatase at 10 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Predicted band size: 48 kDa

    Observed band size: 55 kDa

  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    All lanes: Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (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: 45 kDa, 55 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Purified ab52903 staining Smad3 in Mouse kidney tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraffin and antigen retrieval was by heat mediation using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1/200 dilution. A ready to use rabbit specific IHC polymer detection kit HRP/DAP (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Hematoxylin was used as a counterstain. Nuclear and weakly cytoplasmic staining on mouse kidney without alkaline phosphatase treatment (image A). No signal can be detected when tissues were treated with alkaline phosphatase (image B).

  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    All lanes: Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903) at 1/1000 dilution

    Lane 1: HL-60 (human acute promyelocytic leukemia) treated with TGF-β whole cell lysates, plus Smad3 non-phospho peptide at 10 µg

    Lane 2: HL-60 (human acute promyelocytic leukemia) treated with TGF-β whole cell lysates, plus Smad3 (phospho S423/425) peptide at 10 µg

    Lane 3: HL-60 (human acute promyelocytic leukemia) treated with TGF-β whole cell lysates, plus Smad2 non-phospho peptide at 10 µg

    Lane 4: HL-60 (human acute promyelocytic leukemia) treated with TGF-β whole cell lysates, plus Smad2 (phospho S465/467) peptide at 10 µg

    Lane 5: HL-60 (human acute promyelocytic leukemia) treated with TGF-β whole cell lysates, plus Smad1 non-phospho peptide at 10 µg

    Lane 6: HL-60 (human acute promyelocytic leukemia) treated with TGF-β whole cell lysates, plus Smad1 (phospho S463/465) peptide at 10 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/2000 dilution

    Predicted band size: 48 kDa

    Observed band size: 55 kDa

    Exposure time: 3min

  • Dot Blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Dot Blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Dot blot analysis of human Smad 3 (phospho S423 + S425) phospho peptide (Lane 1), Smad 3 (phospho S423) phospho peptide (Lane 2), Smad 3 (phospho S425) phospho peptide (Lane 3) and Smad 3 non-phospho peptide (Lane 4) labelling Smad 3 (phospho S423 + S425) with ab52903 at a dilution of 1/1000. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) was used as the secondary antibody at a dilution of 1/20,000. Blocking and dilution buffer: 5% NFDM /TBST.

  • Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail
    This image is a courtesy of Aaron Gardner

    Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    All lanes: Western blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903) at 1/1000 dilution

    Lane 1: Lysate prepared from untreated human A549 cells at 20 µg

    Lane 2: Lysate prepared from untreated human A549 cells for 30min at 20 µg

    Lane 3: Lysate prepared from TGF-ß1 cells at 10ng/ml for 30min at 20 µg

    Lane 4: Lysate prepared from TNF-a cells at 20ng/ml for 30min at 20 µg

    Lane 5: Lysate prepared from TGF-ß1 and TNF-a cells at above doses for 30min at 20 µg

    Lane 6: Blank DMEM media at 20 µg

    Secondary

    All lanes: Western blot - Donkey Anti-Rabbit IgG H&L (HRP) (Donkey Anti-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: 1hr

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.

  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail
    This image is courtesy of a customer review submitted by Aaron Gardner.

    Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Phospho Smad3 immunofluorescence staining of A549 cells using rabbit anti-Phospho Smad3 antibody

    ab52903 staining Smad3 (phospho S423 + S425) in 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 for 1 hour at RT. Samples were incubated with primary antibody (1/200 dilution 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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Purified ab52903 staining Smad3 in Human stomach tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraffin and antigen retrieval was by heat mediation using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, Ph9.0). Samples were incubated with primary antibody at a 1/200 dilution. A ready to use rabbit specific IHC polymer detection kit HRP/DAP (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Hematoxylin was used as a counterstain. Nuclear and weakly cytoplasmic staining on human stomach without alkaline phosphatase treatment (image A). No signal can be detected when tissues were treated with alkaline phosphatase (image B).

  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Phospho Smad3 immunofluorescence staining of A549 cells using rabbit anti-Phospho Smad3 antibody

    Immunocytochemistry/Immunofluorescence analysis of A549 +/- TGFβ (5ng/ml 24h) and A549 + TGFβ (5ng/ml 24h) + Lamda phosphatase (LP) cells. Smad3 (phospho S423 + S425) was labelled with purified ab52903 at a dilution of 1/100 dilution while Smad3 was labelled with Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade ab207447 at a dilution of 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (goat anti-rabbit IgG Alexa Fluor® 488) (1/1000 dilution) was used as the secondary antibody. The cells were co-stained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution. Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

  • ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab52903 [EP823Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

  • ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab52903 [EP823Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

  • ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 A549 (Human lung carcinoma cell line) cells treated with hTGF-β1 (7 ng/mL 1 h) and 5 µg of ab52903 [EP823Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

  • Dot Blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903), expandable thumbnail

    Dot Blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903)

    Blocking and diluting buffer and concentration: 5% NFDM /TBST

    All lanes: Dot Blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] (ab52903) at 1/1000 dilution

    Lane 1: Smad5 non-phospho peptide

    Lane 2: Smad5 (s463+s465) phospho peptide

    Lane 3: Smad2 non-phospho peptide

    Lane 4: Smad2 (s465+s467) phospho peptide

    Lane 5: Smad1 non-phospho peptide

    Lane 6: Smad1 (s463+s465) phospho peptide

    Lane 7: Smad3 non-phospho peptide

    Lane 8: Smad3 (s423+s435) phospho peptide

    Secondary

    All lanes: Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Exposure time: 59s

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