Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] KO Tested (ab316117)

Knockout Tested Rabbit Recombinant Monoclonal SMAD2 phospho S465 + S467 antibody. Suitable for Dot, WB, IP, Flow Cyt (Intra), ICC/IF and reacts with Synthetic peptide - Human, Human, Mouse, Rat samples.

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Images

Dot Blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (AB316117), expandable thumbnail

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

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

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Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

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

Tested
Tested

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

Expected
Expected

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

Tested
Tested

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

Not recommended
Not recommended

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

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes -
Species Mouse	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 Human	Dilution info 1/5000	Notes -
Species Mouse	Dilution info 1/5000	Notes -

Not recommended
Not recommended

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

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes -
Species Mouse	Dilution info 1/500	Notes -

Not recommended
Not recommended

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

Not recommended
Not recommended

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

Not recommended
Not recommended

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

Target data

See full target information SMAD2 phospho S465 + 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: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogens: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR28610-44
Purification technique: Affinity purification Protein A
Specificity: Not suitable for rat FC and ICC/IF.
When tested in dot blot this antibody recognizes Smad2 phosphorylated at both S465 and S467 and does not cross-react with Smad2 when singly phosphorylated at S465 or S467.
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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Full details and terms and conditions can be found here:
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9 product images

Dot Blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Dot blot analysis of Smad2 (phospho S465 + S467) using ab316117 at 1:1000 (0.507 ug/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.
The antibody recognizes Smad2 phosphorylated at S465 and S467. It does not cross-react with Smad2 when singly phosphorylated at S465 or S467.
All lanes: Dot Blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117) at 1/1000 dilution
Lane 1: Smad2 (phospho S465+S467) peptide
Lane 2: Smad2 (phospho S465) peptide
Lane 3: Smad2 (phospho S467) peptide
Lane 4: Smad2 non-phospho peptide
Secondary
All lanes: Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Exposure time: 180s
Western blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
The molecular weight observed is consistent with what has been described in the literature (PMID: 34655614).
The up-regulation of pSmad2 is induced by TGF-beta1 treatment (PMID: 34655614, 24347165).
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
All lanes: Western blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117) at 1/1000 dilution
Lane 1: Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg with 5% NFDM/TBST
Lane 2: NIH/3T3 treated with 20 ng/ml TGF beta 1 for 15 minutes whole cell lysate at 20 µg with 5% NFDM/TBST
Lane 3: Untreated C6 (rat glial tumor glial cell) whole cell lysate at 20 µg with 5% NFDM/TBST
Lane 4: C6 treated with 10 ng/ml TGF beta 3 for 30 minutes whole cell lysate at 20 µg with 5% NFDM/TBST
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 60 kDa, 36 kDa
Exposure time: 70s
Western blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
The molecular weight observed is consistent with what has been described in the literature (PMID: 34655614).
The up-regulation of pSmad2 is induced by TGF-beta treatment (PMID: 34655614, 24347165).
In Western blot, ab316117 was shown to bind specifically to Smad2. Target of interest was observed at 60 kDa in wild-type Hela cell lysates (lanes 2) with no signal observed at this size in Smad2 knockout cell line lysates (lanes 3-4 KO cell line Human SMAD2 knockout HeLa cell line ab255430/KO cell lysate Human SMAD2 knockout HeLa cell lysate ab263833 ).
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-Smad2 antibody [EP784Y] - Total protein control (Anti-Smad2 antibody [EP784Y] ab40855) staining at 1/1000 dilution.
All lanes: Western blot - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117) at 1/1000 dilution
Lane 1: Untreated wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg with 5% NFDM/TBST
Lane 2: Wild-type Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (untreated membrane) at 20 µg with 5% NFDM/TBST
Lane 3: Untreated Smad2 knockout Hela whole cell lysate (untreated membrane) at 20 µg with 5% NFDM/TBST
Lane 4: Smad2 knockout Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (untreated membrane) at 20 µg with 5% NFDM/TBST
Lane 5: Untreated wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg with 5% NFDM/TBST
Lane 6: Wild-type Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg with 5% NFDM/TBST
Lane 7: Untreated Smad2 knockout Hela whole cell lysate (alkaline phosphatase treated membrane) at 20 µg with 5% NFDM/TBST
Lane 8: Smad2 knockout Hela treated with 20 ng/ml TGF beta 1 for 60 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg with 5% NFDM/TBST
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Performed under reducing conditions.
Observed band size: 60 kDa, 36 kDa
Exposure time: 37s
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labellingSmad2 (phospho S465 + S467) with ab316117 at 1/500 (1.014 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution.
Confocal image showing nuclear staining in NIH/3T3 cells (shown in green) treated with TGF-β1 (20 ng/mL) for 15 min. The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).
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 (2.5 ug/ml) dilution. The Nuclear counterstain was DAPI.
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SMAD2 KO HeLa (human cervical adenocarcinoma epithelial cell) (Human SMAD2 knockout HeLa cell line ab255430) cells labellingSmad2 (phospho S465 + S467) with ab316117 at 1/500 (1.014 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution.
Confocal image showing nuclear and weak cytoplasmic staining in parental HeLa cells (shown in green) treated with TGF-β1 (20 ng/mL) for 1 hr. The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).
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 (2.5 ug/ml) dilution. The Nuclear counterstain was DAPI.
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.
Flow Cytometry (Intracellular) - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized (SMAD2 KO HeLa (human cervical adenocarcinoma epithelial cell) treated with 20ng/ml TGF-β1 for 1h (Red) and untreated SMAD2 KO HeLa (dotted red)) (Left) / (Parental HeLa treated with 20ng/ml TGF-β1 for 1h (Red) and untreated HeLa (dotted red)) (Right) cells labelling Smad2 (phospho S465 + S467) with ab316117 at 1/5000 dilution (0.01 ug)/Red and Dotted 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) preadsorbed ab150081) at 1/5000 dilution was used as the secondary antibody.
Immunoprecipitation - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Smad2 (phospho S465 + S467) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate with ab316117 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316117 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate

Lane 2: ab316117 IP in NIH/3T3 (mouse embryonic fibroblast) 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 ab316117 in NIH/3T3 treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate
All lanes: Immunoprecipitation - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117) at 1/30 dilution
All lanes: NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate with 5% NFDM/TBST
Secondary
All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution
Exposure time: 180s
Immunoprecipitation - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Smad2 (phospho S465 + S467) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 60 minutes whole cell lysate with ab316117 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316117 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 60 minutes, whole cell lysate

Lane 2: ab316117 IP in HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 60 minutes whole cell lysate

Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab316117 in HeLa treated with 20ng/ml TGF beta1 for 60 minutes whole cell lysate
All lanes: Immunoprecipitation - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117) at 1/30 dilution
All lanes: HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 60 minutes whole cell lysate with 5% NFDM/TBST
Secondary
All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution
Exposure time: 58s
Flow Cytometry (Intracellular) - Anti-Smad2 (phospho S465 + S467) antibody [EPR28610-44] (ab316117)
Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta 1 for 15 min (Red) / untreated NIH/3T3 (dotted red) cells labelling Smad2 (phospho S465 + S467) with ab316117 at 1/5000 dilution (0.01 ug)/Red and Dotted 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) preadsorbed ab150081) at 1/5000 dilution was used as the secondary antibody.