Product: Phospho-Smad2 (Ser467) Antibody
Catalog: AF3449
Source: Rabbit
Application: WB, IHC, IF/ICC, ELISA(peptide)
Reactivity: Human, Mouse, Rat
Prediction: Pig, Zebrafish, Bovine, Sheep, Rabbit, Dog, Xenopus
Mol.Wt.: 58kD; 52kD(Calculated).
Uniprot: Q15796
RRID: AB_2834844

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500, ELISA(peptide) 1:20000-1:40000
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Prediction:
Pig(100%), Zebrafish(100%), Bovine(100%), Sheep(100%), Rabbit(100%), Dog(100%), Xenopus(100%)
Clonality:
Polyclonal
Specificity:
Phospho-Smad2 (Ser467) Antibody detects endogenous levels of Smad2 only when phosphorylated at Serine 467.
RRID:
AB_2834844
Cite Format: Affinity Biosciences Cat# AF3449, RRID:AB_2834844.
Conjugate:
Unconjugated.
Purification:
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

Drosophila, homolog of, MADR2; hMAD-2; HsMAD2; JV18; JV18-1; JV181; MAD; MAD homolog 2; MAD Related Protein 2; Mad-related protein 2; MADH2; MADR2; MGC22139; MGC34440; Mother against DPP homolog 2; Mothers against decapentaplegic homolog 2; Mothers against decapentaplegic, Drosophila, homolog of, 2; Mothers against DPP homolog 2; OTTHUMP00000163489; Sma and Mad related protein 2; Sma- and Mad-related protein 2 MAD; SMAD 2; SMAD family member 2; SMAD, mothers against DPP homolog 2; SMAD2; SMAD2_HUMAN;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Expression:
Q15796 SMAD2_HUMAN:

Expressed at high levels in skeletal muscle, endothelial cells, heart and placenta.

Description:
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.
Sequence:
MSSILPFTPPVVKRLLGWKKSAGGSGGAGGGEQNGQEEKWCEKAVKSLVKKLKKTGRLDELEKAITTQNCNTKCVTIPSTCSEIWGLSTPNTIDQWDTTGLYSFSEQTRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEVCVNPYHYQRVETPVLPPVLVPRHTEILTELPPLDDYTHSIPENTNFPAGIEPQSNYIPETPPPGYISEDGETSDQQLNQSMDTGSPAELSPTTLSPVNHSLDLQPVTYSEPAFWCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSVRCSSMS

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Pig
100
Bovine
100
Sheep
100
Dog
100
Xenopus
100
Zebrafish
100
Rabbit
100
Horse
0
Chicken
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - Q15796 As Substrate

Site PTM Type Enzyme
Ubiquitination
S2 Acetylation
S2 Phosphorylation
T8 Phosphorylation P27361 (MAPK3) , P24941 (CDK2)
K13 Ubiquitination
K19 Acetylation
K20 Acetylation
S21 Phosphorylation
K39 Acetylation
K46 Sumoylation
S47 Phosphorylation
K63 Ubiquitination
Y102 Phosphorylation
S110 Phosphorylation Q9UQM7 (CAMK2A) , Q9H4A3 (WNK1)
S118 Phosphorylation
K121 Ubiquitination
Y128 Phosphorylation
K156 Sumoylation
K156 Ubiquitination
K157 Ubiquitination
Y165 Phosphorylation
T172 Phosphorylation
T197 Phosphorylation P25098 (GRK2)
T220 Phosphorylation P28482 (MAPK1) , P27361 (MAPK3)
S240 Phosphorylation Q9UQM7 (CAMK2A)
S245 Phosphorylation Q14680 (MELK) , P28482 (MAPK1) , P27361 (MAPK3)
S250 Phosphorylation P28482 (MAPK1) , P27361 (MAPK3)
S255 Phosphorylation P28482 (MAPK1) , P27361 (MAPK3)
S260 Phosphorylation Q9UQM7 (CAMK2A) , Q9H4A3 (WNK1)
S317 Phosphorylation
T324 Phosphorylation
S417 Phosphorylation Q13177 (PAK2)
K420 Acetylation
S458 Phosphorylation
S460 Phosphorylation
S464 Phosphorylation P36897 (TGFBR1) , O00238 (BMPR1B)
S465 Phosphorylation Q9H4A3 (WNK1) , P36897 (TGFBR1) , O00238 (BMPR1B) , O96013 (PAK4) , Q8NER5 (ACVR1C)
S467 Phosphorylation O00238 (BMPR1B) , P36897 (TGFBR1) , Q8NER5 (ACVR1C)

Research Backgrounds

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.

PTMs:

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. TGF-beta-induced Ser-465/467 phosphorylation declines progressively in a KMT5A-dependent manner. 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-240 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. Monoubiquitinated, leading to prevent DNA-binding (By similarity). Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes. Ubiquitinated by RNF111, leading to its degradation: only SMAD2 proteins that are 'in use' are targeted by RNF111, RNF111 playing a key role in activating SMAD2 and regulating its turnover (By similarity).

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.

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 (PubMed:9865696). On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1 (PubMed:16751101, PubMed:19289081).

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Expressed at high levels in skeletal muscle, endothelial cells, heart and placenta.

Subunit Structure:

Monomer; 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 (By similarity). Interacts with NEDD4L in response to TGF-beta (By similarity). 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, SKI and SNW1. 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 C-terminal R domain); the interaction results in the export of dephosphorylated SMAD3 out of the nucleus and termination of the TGF-beta signaling. Interacts with PDPK1 (via PH domain). Interacts with DAB2; the interactions are enhanced upon TGF-beta stimulation. Interacts with USP15. Interacts with PPP5C. Interacts with ZNF580. Interacts with LDLRAD4 (via the SMAD interaction motif). Interacts (via MH2 domain) with PMEPA1 (via the SMAD interaction motif). Interacts with ZFHX3. Interacts with ZNF451. Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4. Interacts weakly with ZNF8 (By similarity). Interacts (when phosphorylated) with RNF111; RNF111 acts as an enhancer of the transcriptional responses by mediating ubiquitination and degradation of SMAD2 inhibitors (By similarity).

Family&Domains:

Belongs to the dwarfin/SMAD family.

Research Fields

· Cellular Processes > Cell growth and death > Cell cycle.   (View pathway)

· Cellular Processes > Transport and catabolism > Endocytosis.   (View pathway)

· Cellular Processes > Cell growth and death > Cellular senescence.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Adherens junction.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Signaling pathways regulating pluripotency of stem cells.   (View pathway)

· Environmental Information Processing > Signal transduction > FoxO signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > TGF-beta signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Apelin signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Hippo signaling pathway.   (View pathway)

· Human Diseases > Infectious diseases: Parasitic > Chagas disease (American trypanosomiasis).

· Human Diseases > Infectious diseases: Viral > HTLV-I infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Proteoglycans in cancer.

· Human Diseases > Cancers: Specific types > Colorectal cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Pancreatic cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Hepatocellular carcinoma.   (View pathway)

· Human Diseases > Cancers: Specific types > Gastric cancer.   (View pathway)

· Human Diseases > Immune diseases > Inflammatory bowel disease (IBD).

· Organismal Systems > Immune system > Th17 cell differentiation.   (View pathway)

· Organismal Systems > Endocrine system > Relaxin signaling pathway.

References

1). Ding D et al. Timosaponin BII inhibits TGF‐β mediated epithelial‐mesenchymal transition through Smad‐dependent pathway during pulmonary fibrosis. Phytother Res 2023 Feb 18. (PubMed: 36807664) [IF=6.388]

2). Liu H et al. Enhancer of zeste homolog 2 modulates oxidative stress-mediated pyroptosis in vitro and in a mouse kidney ischemia-reperfusion injury model. FASEB J 2020 Jan;34(1):835-852 (PubMed: 31914694) [IF=5.834]

Application: WB    Species: human    Sample: HK-2 cells

FIGURE 7|EZH2 regulated Nox4 expression via the ALK5/Smad2/Smad3 pathway. D-G, HK-2 cells were transfected with an siRNA against EZH2 or a negative control siRNA (si-NC) for 48 h before being exposed to H/R. D-F, Western blot analysis for the protein expression of ALK5, Smad2, Smad3, p-Smad2, and Smad3 in the indicated groups and quantitative analysis of ALK5, p-Smad2, and p-Smad3, n = 3.

3). Dong M et al. Dietary protein levels changed the hardness of muscle by acting on muscle fiber growth and the metabolism of collagen in sub-adult grass carp (Ctenopharyngodon idella). J Anim Sci Biotechnol 2022 Aug 25;13(1):109. (PubMed: 36002862) [IF=5.032]

4). Li X et al. Regorafenib-Attenuated, Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway. Int J Mol Sci 2021 Feb 17;22(4):1985. (PubMed: 33671452) [IF=4.556]

Application: WB    Species: Mouse    Sample: Mlg cells

Figure 3. Regorafenib down-regulates TGF-β1/Smad and TGF-β1/non-Smad signals in pulmonary fibroblasts. (A) CAGAmouse embryonic fibroblast (NIH-3T3) cells were exposed to TGF-β1 (5 ng/mL) or a series concentration (0–32 µM) in serum-free medium for 18 h; (B) Mlg cells were treated with TGF-β1 (5 ng/mL) and/or RG (2 µM, 4 µM) for 30 min, and Western blot was used to detect Smad3, Smad2, and their phosphorylation expression levels. Densitometric analysis are shown beside; (C) Mlg cells were incubated with RG (2 µM, 4 µM) and/or TGF-β1 (5 ng/mL) for 12 h to analyze the Erk1/2 and Akt and their phosphorylation levels by Western blotting. Densitometric analysis are shown beside; (D) BLM-PPF cells were incubated with RG (2 µM, 4 µM) for 12 h to analyze Erk1/2 and Akt and their phosphorylation levels by Western blotting. Densitometric analysis are shown beside. β-tubulin or GAPDH were used as a loading control. Data in (A–D) are means ± standard error of mean (SEM); ### p < 0.001, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA), NS: not significant.

5). Gu S et al. Retinal pigment epithelial cells secrete miR-202-5p-containing exosomes to protect against proliferative diabetic retinopathy. Exp Eye Res 2020 Sep 29;108271. (PubMed: 33007305) [IF=3.770]

Application: WB    Species: human    Sample: ARPE19 cells

Figure 4. |miR-202-5p is highly expressed in high glucose (HG)-treated ARPE19 cells and participates in the TGF/Smad pathway.(C) Western blotting was used to analyze the expression of the TGF/Smad pathway proteins. Phosphorylation of Smad2 protein was reduced after the treatment of ARPE19-derived exosomes in the HG group.

6). Yang C et al. Iodine-125 Seeds Inhibit Carcinogenesis of Hepatocellular Carcinoma Cells by Suppressing Epithelial-Mesenchymal Transition via TGF-β1/Smad Signaling. Dis Markers 2022 May 7;2022:9230647. (PubMed: 35578690) [IF=3.464]

7). Xu H et al. Effects of Ziyin Qianyang Formula on Renal Fibrosis through the TGF-β1/Smads Signaling Pathway in Spontaneously Hypertensive Rats. Evid Based Complement Alternat Med 2022 Nov 7;2022:6088673. (PubMed: 36387359) [IF=2.650]

8). Fang CC et al. Effects of dietary methionine on growth performance, muscle nutritive deposition, muscle fibre growth and type I collagen synthesis of on-growing grass carp (Ctenopharyngodon idella). Br J Nutr 2020 Jul 28;1-36. (PubMed: 32718370)

Application: WB    Species: grass carp    Sample: muscle

Fig. 7. |Western blot analysis of protein expression of genes involved in type I collagen metabolism in the muscle of on-growing grass carp (Ctenopharyngodon idella) fed diets with graded levels of Met(g kg -1) for 60 days. Data represent means of three fish in each group, error bars indicate S.D. Values having different letters are significantly different

9). Guan G et al. TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial-mesenchymal transition in diabetic nephropathy. J Biol Chem 2022 Feb 11;101725. (PubMed: 35157852)

Application: WB    Species: Human    Sample: hRGECs

Figure 9 TFPI2 promotes TGF-β/Smad signaling activation. Human renal glomerular endothelial cells (hRGECs) were infected with adenovirus encoding shRNA targeting TFPI2 (TFPI2 shRNA) or overexpressing TFPI2 (TFPI2 OE), followed by stimulation of 5 ng/ml TGF-β2 for 48 h. A, the expression of SMAD7, TGFBR1, TGFBR2, SMAD2/3, and phospho-SMAD2/3 (p-SMAD2/3) was determined by Western blot. Semiquantitative analysis of (B) SMAD7, (C) TGFBR1, and (D) TGFBR2, as well as (E and F) the ratio of p-SMAD2/3 to SMAD2/3. G and H, immunofluorescent staining of SMAD2/3 in hRGECs. Yellow arrows indicated nuclear translocation of SMAD2/3. Data are shown as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. TGF-β, transforming growth factor beta; TFP12, tissue factor pathway inhibitor 2.

10). Flavonoids of Coreopsis tinctoria Nutt alleviate the oxidative stress and inflammation of glomerular mesangial cells in diabetic nephropathy via RhoA/ROCK signaling.

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