Rabbit Anti-Phospho-Smad2 (Ser245 + Ser250 + Ser255) antibody

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phospho-Smad2(p-SerSer245/250/255); p-Smad2(Ser245/250/255); phospho-Smad2(p-Ser245/250/255); hMAD 2; hSMAD2; JV18 1; JV18; JV181; MAD; MAD Related Protein 2; MADH2; MADR2; MGC22139; MGC34440; Mothers Against Decapentaplegic Homolog 2; mothers against DPP

Cat:

SL3420R

Species Reactivity：

Human,Mouse,(predicted: Rat,Dog,Pig,Cow,)

Immunogen：

KLH conjugated Synthesised phosphopeptide derived from human Smad2 around the phosphorylation site of Ser245/250/255:TG(p-S)PAEL(p-S)PTTL(p-S)PV

Format：

Liquid

Storage instructions：

Shipped at 4℃. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles.

Concentration：

1mg/ml

Clonality：

Polyclonal

Isotype：

IgG

Applications：

WB=1:500-2000ELISA=1:5000-10000IHC-P=1:100-500IHC-F=1:100-500Flow-Cyt=3μg/TestIF=1:100-500（Paraffin sections need to do antigen repair）not yet tested in other applications.optimal dilutions/concentrations should be determined by the end user.

Host：

Rabbit

Product Overview：

Sample: MCF 7 (Human) Lysate at 30 ug Primary: Anti-Phospho-Smad2(Ser245+Ser250+Ser255)(SL3420R) at 1/300 dilutionSecondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilutionPredicted band size: 58 kDObserved band size: 58 kDParaformaldehyde-fixed, paraffin embedded (Mouse placenta); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-Smad2(Ser245 + Ser250 + Ser255)) Polyclonal Antibody, Unconjugated (SL3420R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-Smad2(Ser245 + Ser250 + Ser255)) Polyclonal Antibody, Unconjugated (SL3420R ) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining. Blank control (Black line): Mouse spleen (Black). Primary Antibody (green line): Rabbit Anti-Phospho-Smad2(Ser245 + Ser250 + Ser255)antibody (SL3420R) Dilution: 3μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-PEDilution: 1μg /test. ProtocolThe cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at room temperature. The cells were then incubated in 5%BSA goat serum to block non-specific protein-protein interactions for 15 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

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Unit:

Price: $256.00

Product PDFs

Datasheet: Down Datasheet

Other Information
Citations
Protein Attributes
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The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is recruited to the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to TGF-beta signal, this protein is phosphorylated by the TGF-beta receptors. The phosphorylation induces the dissociation of this protein with SARA and the association with the family member SMAD4. The association with SMAD4 is important for the translocation of this protein into the nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. This protein can also be phosphorylated by activin type 1 receptor kinase, and mediates the signal from the activin. Alternatively spliced transcript variants have been observed for this gene. [provided by RefSeq, May 2012]

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. May act as a tumor suppressor in colorectal carcinoma. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.

Subunit:
Momomer; the absence of TGF-beta. Heterodimer; in the presence of TGF-beta. Forms a heterodimer with co-SMAD, SMAD4, in the nucleus to form the transactivation complex SMAD2/SMAD4. Interacts with AIP1, HGS, PML and WWP1. Interacts with NEDD4L in response to TGF-beta. Found in a complex with SMAD3 and TRIM33 upon addition of TGF-beta. Interacts with ACVR1B, SMAD3 and TRIM33. Interacts (via the MH2 domain) with ZFYVE9; may form trimers with the SMAD4 co-SMAD. Interacts with FOXH1, homeobox protein TGIF, PEBP2-alpha subunit, CREB-binding protein (CBP), EP300 and SKI. Interacts with SNON; when phosphorylated at Ser-465/467. Interacts with SKOR1 and SKOR2. Interacts with PRDM16. Interacts (via MH2 domain) with LEMD3. Interacts with RBPMS. Interacts with WWP1. Interacts (dephosphorylated form, via the MH1 and MH2 domains) with RANBP3 (via its SLCterminal R domain); the interaction results in the export of dephosphorylated SMAD3 out of the nucleus and termination ot the TGF-beta signaling. Interacts with PDPK1 (via PH domain).

Subcellular Location:
Cytoplasm. Nucleus. Note=Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1.

Tissue Specificity:
Expressed at high levels in skeletal muscle, heart and placenta.

Post-translational modifications:
Phosphorylated on one or several of Thr-220, Ser-245, Ser-250, and Ser-255. In response to TGF-beta, phosphorylated on Ser-465/467 by TGF-beta and activin type 1 receptor kinases. Able to interact with SMURF2 when phosphorylated on Ser-465/467, recruiting other proteins, such as SNON, for degradation. In response to decorin, the naturally occurring inhibitor of TGF-beta signaling, phosphorylated on Ser-48 by CaMK2. Phosphorylated by MAPK3 upon EGF stimulation; which increases transcriptional activity and stability, and is blocked by calmodulin. Phosphorylated by PDPK1.
In response to TGF-beta, ubiquitinated by NEDD4L; which promotes its degradation.
Acetylated on Lys-19 by coactivators in response to TGF-beta signaling, which increases transcriptional activity. Isoform short: Acetylation increases DNA binding activity in vitro and enhances its association with target promoters in vivo. Acetylation in the nucleus by EP300 is enhanced by TGF-beta.

Similarity:
Belongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain.

SWISS:
Q15796

Gene ID:
4087

Database links:

Entrez Gene: 4087 Human

Entrez Gene: 17126 Mouse

Entrez Gene: 29357 Rat

Omim: 601366 Human

SwissProt: Q15796 Human

SwissProt: Q62432 Mouse

SwissProt: O70436 Rat

Unigene: 12253 Human

Unigene: 705764 Human

Unigene: 391091 Mouse

Unigene: 2755 Rat

Picture

Paraformaldehyde-fixed, paraffin embedded (Mouse placenta); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-Smad2(Ser245 + Ser250 + Ser255)) Polyclonal Antibody, Unconjugated (SL3420R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-Smad2(Ser245 + Ser250 + Ser255)) Polyclonal Antibody, Unconjugated (SL3420R ) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Blank control (Black line): Mouse spleen (Black).
Primary Antibody (green line): Rabbit Anti-Phospho-Smad2(Ser245 + Ser250 + Ser255)antibody (SL3420R)
Dilution: 3μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at room temperature. The cells were then incubated in 5%BSA goat serum to block non-specific protein-protein interactions for 15 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

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