Name: Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free
SKU: ab172202
Rating: 3 (1 reviews)

Rabbit Recombinant Monoclonal SMAD3 phospho S423 + S425 antibody. Carrier free. Suitable for Dot, WB, ICC/IF, IHC-P, ChIC/CUT&RUN-seq and reacts with Human, Mouse samples. Cited in 1 publication.

Related conjugates and formulations

ab311071
Conjugated
Alexa Fluor® 647 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]
ab312931
Conjugated
Alexa Fluor® 568 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]
ab52903
Unconjugated
Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]
ab310954
Conjugated
Alexa Fluor® 488 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]
ab321670
Conjugated
Alexa Fluor® 750 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]
ab313143
Conjugated
Alexa Fluor® 555 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]
ab311658
Conjugated
Alexa Fluor® 594 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y]

Images

Immunocytochemistry/ Immunofluorescence - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free (AB172202), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Constituents: PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

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

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	Dot	WB	ICC/IF	IHC-P	ChIC/CUT&RUN-seq	IP	Flow Cyt
Human	Tested	Tested	Tested	Tested	Tested	Not recommended	Not recommended
Mouse	Expected	Tested	Expected	Tested	Expected	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse, Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Mouse	Dilution info -	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	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse, Human	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse, Human	Dilution info -	Notes -

Associated Products

Select an associated product type

15 products for Alternative Product

2 products for Alternative Version

Target data

See full target information SMAD3 phospho S423 + S425

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.

Additional Targets

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

Alternative names

MADH3, SMAD3, Mothers against decapentaplegic homolog 3, MAD homolog 3, Mad3, Mothers against DPP homolog 3, hMAD-3, JV15-2, SMAD family member 3, SMAD 3, Smad3, hSMAD3

Recommended products

Rabbit Recombinant Monoclonal SMAD3 phospho S423 + S425 antibody. Carrier free. Suitable for Dot, WB, ICC/IF, IHC-P, ChIC/CUT&RUN-seq and reacts with Human, Mouse samples. Cited in 1 publication.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Constituents: PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Carrier free: Yes
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

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: +4°C
Storage information: Do Not Freeze

Notes

ab172202 is the carrier-free version of Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903.

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.

Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.

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 that plays a mechanical role in signal transduction. It acts mainly as a transcription factor and gets activated through phosphorylation. The molecular weight of Smad3 is approximately 48 kDa. It is expressed widely across numerous tissues including the cellular nucleus where it executes its function after activation.

Biological function summary

Smad3 acts as a mediator of signal transduction for the TGF-beta (transforming growth factor-beta) superfamily forming a complex with phosphorylated Smad2. This enables it to regulate transcriptional activity influencing cell proliferation differentiation and apoptosis. Smad3 also participates in various cellular processes by interacting with other co-factors and regulatory proteins that aid in fine-tuning its function.

Pathways

Smad3 plays an important role in the TGF-beta signaling pathway where it works closely with Smad4 to propagate the signal. Upon phosphorylation it forms a complex with co-Smad (Smad4) and moves into the nucleus to influence gene expression. Smad3 is also involved in pathways related to oncogenesis and tissue fibrosis indicating its significant role in cellular regulation and response mechanisms.

Associated diseases and disorders

Smad3 is associated with fibrotic diseases and cancers particularly in tissues such as the liver and lungs. Altered Smad3 signaling contributes to the pathological process occurring in fibrotic disorders often interacting with Smad4 in these abnormalities. Dysregulated Smad3 expression or mutations can also lead to oncogenic transformations highlighting its critical involvement in disease states.

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.

13 product images

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] - BSA and Azide free (ab172202)
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 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903) in CTL-siRNA treated MEFs (B) while only weak nuclear staining for MEFs treated with NDRG1-siRNA (D).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
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] - BSA and Azide free (ab172202)
Phospho Smad3 immunofluorescence staining of primary embyronic epicardial cells using rabbit anti-Phospho Smad3 antibody

Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
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] - BSA and Azide free (ab172202)
Representative IHC photomicrographs from an Environmental enteropathy (EE) duodenal biopsy showing p-SMAD3 staining (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903) in only the epithelium (arrows).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free (ab172202)
Purified Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
Dot Blot - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free (ab172202)
Dot blot analysis of 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 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free (ab172202)
Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903 at 1/100 dilution.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
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] - BSA and Azide free (ab172202)
Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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: 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free (ab172202)
This IHC data was generated using the same anti-phospho Smad3 S423/425 antibody clone, EP823Y, in a different buffer formulation (cat# Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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] - BSA and Azide free (ab172202)
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 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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.
This ICC/IF data was generated using the same anti-phospho Smad3 S423/425 antibody clone, EP823Y, in a different buffer formulation (Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] ab52903).
ChIC/CUT&RUN sequencing - Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] - BSA and Azide free (ab172202)
This data was developed using the same antibody clone in a different buffer formulation (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 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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] - BSA and Azide free (ab172202)
This data was developed using the same antibody clone in a different buffer formulation (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 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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] - BSA and Azide free (ab172202)
This data was developed using the same antibody clone in a different buffer formulation (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 Anti-SMAD3 (pS423/425) + SMAD5 (pS463/465 ) + SMAD1 (pS463/465) + SMAD2 (pS465/467) antibody [EP823Y] 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] - BSA and Azide free (ab172202)
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] (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