Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)

Rabbit Recombinant Monoclonal SMAD2 phospho S467 antibody. Suitable for ICC/IF, IP, Dot, WB, Flow Cyt (Intra), IHC-P and reacts with Mouse, Human, Rat, Synthetic peptide - Human samples. Cited in 22 publications.

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Alexa Fluor® 488 Anti-Smad2 (phospho S467) antibody [EPR23681-40]

Images

Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (AB280888), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Preservative: 0.01% Sodium azide
Constituents: 59.94% 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

	ICC/IF	IP	Dot	WB	Flow Cyt (Intra)	IHC-P
Human	Tested	Tested	Expected	Tested	Tested	Tested
Mouse	Tested	Tested	Expected	Tested	Tested	Tested
Rat	Expected	Expected	Expected	Not recommended	Expected	Tested
Synthetic peptide - Human	Not recommended	Not recommended	Tested	Not recommended	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/50	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/50	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/30	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/30	Notes -

Expected
Expected

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info 1/1000	Notes -

Expected
Expected

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

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000	Notes -
Species Human	Dilution info 1/1000	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Rat, Synthetic peptide - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/5000	Notes -
Species Human	Dilution info 1/5000	Notes -

Expected
Expected

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

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info -	Notes -

Target data

See full target information SMAD2 phospho S467

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 SMAD2/SMAD4 complex, activates transcription. Promotes TGFB1-mediated transcription of odontoblastic differentiation genes in dental papilla cells (By similarity). Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator. May act as a tumor suppressor in colorectal carcinoma (PubMed:8752209).

Alternative names

MADH2, MADR2, SMAD2, Mothers against decapentaplegic homolog 2, MAD homolog 2, Mothers against DPP homolog 2, JV18-1, Mad-related protein 2, SMAD family member 2, hMAD-2, SMAD 2, Smad2, hSMAD2

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR23681-40
Purification technique: Affinity purification Protein A
Specificity: This antibody was raised against a dual phosphorated peptide for both phosopho S467 and phosopho S465. The dot blot shows that this antibody binds phosopho S467 on its own, but binding is enhanced by the presence of phosopho S465. Our data suggests shown in the dot blot that this antibody does not bind phosopho S465 alone.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
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.

Supplementary info

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

Activity summary

Smad2 also known as Mothers against decapentaplegic homolog 2 (MAD2) or MADR2 is a signaling protein involved in the transforming growth factor-beta (TGF-β) receptor pathway. Smad2 has a molecular mass of approximately 58 kDa and expresses in various tissues including epithelial mesenchymal and endothelial cells. Smad2 undergoes phosphorylation on serine residues in response to TGF-β signaling converting it into phosphorylated forms often referred to as p-Smad2 or phospho-Smad2. These phosphorylated forms are critical for the relay of signals from the cell surface to the nucleus.

Biological function summary

Smad2 acts as an intracellular mediator for TGF-β signaling a pathway important for regulating cell proliferation differentiation and apoptosis. Smad2 typically functions as part of a heteromeric complex with Smad4 another key player in TGF-β signaling. Upon activation phosphorylated Smad2 combines with Smad4 to form a complex that translocates into the nucleus. This complex then binds to specific DNA sequences to regulate the transcription of target genes involved in processes such as cell growth inhibition and extracellular matrix production.

Pathways

Smad2 is integral to the TGF-β and activin receptor signaling pathways. These pathways are essential in controlling cell growth and immune responses. Smad2 interacts with other proteins such as Smad3 in addition to Smad4 to modulate gene expression effectively. The interaction between Smad2 and these proteins ensures precise control of cellular responses to external stimuli emphasizing its pivotal role in maintaining cellular homeostasis.

Associated diseases and disorders

Smad2 correlates with various pathological conditions including fibrosis and cancer. Aberrant Smad2 signaling can contribute to the development of these diseases as excessive TGF-β signaling promotes fibrotic tissue deposition and tumor progression. Smad2 connects with other proteins like Smad3 in these pathological contexts both acting as mediators of abnormal cellular behaviors. Understanding the regulatory mechanisms of Smad2 can help develop therapeutic strategies against disorders linked to dysregulated TGF-β signaling.

Product promise

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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:
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15 product images

Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lanes 1-4: Merged signal (red and green). Green - ab280888 observed at 60 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
ab280888 Anti-Smad2 (phospho S467) antibody [EPR23681-40] was shown to specifically react with Smad2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human SMAD2 knockout HeLa cell line ab255430 (knockout cell lysate Human SMAD2 knockout HeLa cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE.
ab280888 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated at 4℃ overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1: Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2: Wild-type HeLa (human cervix adenocarcinoma epithelial cell) treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 µg
Lane 2: Western blot - Human SMAD2 knockout HeLa cell line (Human SMAD2 knockout HeLa cell line ab255430)
Lane 3: Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 4: Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Predicted band size: 52 kDa
Observed band size: 60 kDa
Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lanes 1-4: Merged signal (red and green). Green - ab280888 observed at 60 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
ab280888 Anti-Smad2 (phospho S467) antibody [EPR23681-40] was shown to specifically react with Smad2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human SMAD2 knockout HeLa cell line ab255430 (knockout cell lysate Human SMAD2 knockout HeLa cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE.
ab280888 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated at 4℃ overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1: Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2: Wild-type HeLa (human cervix adenocarcinoma epithelial cell) treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 µg
Lane 3: Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 4: Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Predicted band size: 52 kDa
Observed band size: 60 kDa
Dot Blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Dot blot analysis of Smad2 (phospho S467) using ab280888 at 1:1000 (0.524 μg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1:100,000 dilution.
Lane 1: Smad2 peptide (aa 458-467)
Lane 2: Smad2 (phospho S465) peptide (aa 458-467)
Lane 3: Smad2 (phospho aa S465/S467) peptide (aa 458-467)
Lane 4: Smad2 (phospho S467) peptide (aa 458-467)
Exposure time: 3 minutes
Blocking and diluting buffer and concentration: 5% NFDM/TBST
Immunoprecipitation - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Smad2 (phospho S467) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate 10 μg with ab280888 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280888 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: HeLa treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate 10 μg
Lane 2: ab280888 IP in HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab280888 in HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.
All lanes: Immunoprecipitation - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Predicted band size: 52 kDa
Observed band size: 60 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded human stomach tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on human stomach without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling Smad2 (phospho S467) with ab280888 at 1/50 (10.48 μg/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).
Confocal image showing mainly nuclear staining in HeLa cells treated with TGF-β1 (20 ng/ml) for 15 mins.
Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
Flow Cytometry (Intracellular) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes (Red)/ Untreated control (Green) cells labelling Smad2 (phospho S467) with ab280888 at 1/5000 dilution (0.01μg)/ red (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in HeLa (PMID:24959295).
Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
The molecular weight observed is consistent with what has been described in the literature. (PMID: 24959295）

The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in HeLa (PMID:24959295).
Exposure time: 3 minutes
All lanes: Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate at 10 µg
Lane 2: HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate (phosphatase treated membrane) at 10 µg
Lane 3: HeLa whole cell lysate at 20 µg
Lane 4: HeLa treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 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: 52 kDa
Observed band size: 60 kDa
Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
The molecular weight observed is consistent with what has been described in the literature. (PMID: 24347165)
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in NIH/3T3 (PMID: 24347165)
Exposure time: 3 minutes
All lanes: Western blot - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1: NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 and 50 μM MG-132 for 15 minutes, whole cell lysate at 10 µg
Lane 2: NIH/3T3 treated with 20ng/ml TGF beta1 and 50 μM MG-132 for 15 minutes whole cell lysate (phosphatase treated membrane) at 10 µg
Lane 3: NIH/3T3 whole cell lysate at 10 µg
Lane 4: NIH/3T3 treated with 20ng/ml TGF beta1 and 50 μM MG-132 for 15 minutes, whole cell lysate 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: 52 kDa
Observed band size: 60 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 μg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on human colon carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 μg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on mouse cardiac muscle without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunoprecipitation - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Smad2 (phospho S467) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 50 μM MG-132 for 15 minutes, whole cell lysate 10 μg with ab280888 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280888 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: NIH/3T3 treated with 20ng/ml TGF beta1 50 μM MG-132 for 15 minutes, whole cell lysate 10 μg
Lane 2: ab280888 IP in NIH/3T3 treated with 20ng/mltreated with 20ng/ml TGF beta1 50 μM MG-132 for 15 minutes, whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab280888 in NIH/3T3 treated with 20ng/mltreated with 20ng/ml TGF beta1 50 μM MG-132 for 15 minutes, whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds.
All lanes: Immunoprecipitation - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Predicted band size: 52 kDa
Observed band size: 60 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded rat lung tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 μg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on rat lung without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling Smad2 (phospho S467) with ab280888 at 1/50 (10.48 μg/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).
Confocal image showing mainly nuclear staining in NIH/3T3 cells treated with TGF-β1 (20 ng/ml) for 15 mins.
Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
Flow Cytometry (Intracellular) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 for 15 minutes (Red)/ Untreated control (Green) cells labelling Smad2 (phospho S467) with ab280888 at 1/5000 dilution (0.01μg)/ red (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in NIH/3T3 (PMID: 24347165).