Product: Phospho-Smad2 (Ser250) Antibody
Catalog: AF3450
Description: Rabbit polyclonal antibody to Phospho-Smad2 (Ser250)
Application: WB IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Zebrafish, Bovine, Horse, Sheep, Rabbit, Dog, Xenopus
Mol.Wt.: 65kDa; 52kD(Calculated).
Uniprot: Q15796
RRID: AB_2834845

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Product Info

WB 1:500-1:2000, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.

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.

Zebrafish(86%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Xenopus(86%)
Phospho-Smad2 (Ser250) Antibody detects endogenous levels of Smad2 only when phosphorylated at Serine 250.
Cite Format: Affinity Biosciences Cat# AF3450, RRID:AB_2834845.
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
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.


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;



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

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.



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.

Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - Q15796 As Substrate

Site PTM Type Enzyme
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


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.


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).


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.


1). Histone deacetylase 3 aberration inhibits Klotho transcription and promotes renal fibrosis. Cell Death & Differentiation, 2020 [IF=12.4]

Application: WB    Species: Mouse    Sample: renal tissues

Fig. 6: Klotho derepression is essential for the antifibrosis function of HDAC3 inhibition.Mice receiving siRNA-Control (siCon) or siRNA-Klotho (siKL) were subgrouped into Sham, RGFP966, UUO, and RGFP966-treated UUO (n = 6). a Representative photomicrographs of kidney sections (Masson’s trichrome staining). The dark arrows indicate fibrotic areas. b Quantitation of renal fibrosis in a. The effects of siRNA-Klotho (P1), RGFP966 intervention (P2), and the interaction between siRNA-Klotho and RGFP966 intervention (P3) were indicated. c Western blots. The renal tissues from experimental mice in a were assayed for Klotho, α-SMA, BMP-7, collagen 1 (Colla 1), and d β-catenin (β-cat) and phosphorylated Smad2 (p-Smd2). Two samples from each group were shown. e Quantification of c and d. Data were presented as means ± SEM. *P 

2). Squalene epoxidase promotes hepatocellular carcinoma development by activating STRAP transcription and TGF-β/SMAD signalling. British Journal of Pharmacology, 2023 (PubMed: 36581319) [IF=7.3]

3). L-Glutamine alleviates osteoarthritis by regulating lncRNA-NKILA expression through the TGF-β1/SMAD2/3 signalling pathway. Clinical Science, 2022 (PubMed: 35730575) [IF=6.0]

4). Myricetin ameliorates bleomycin-induced pulmonary fibrosis in mice by inhibiting TGF-β signaling via targeting HSP90β. BIOCHEMICAL PHARMACOLOGY, 2020 (PubMed: 32535102) [IF=5.8]

5). Protective effect of remdesivir against pulmonary fibrosis in mice. Frontiers in Pharmacology, 2021 (PubMed: 34512328) [IF=5.6]

Application: WB    Species: Mice    Sample: lung tissues

FIGURE 10 Remdesivir suppress BLM-induced pulmonary fibrosis in mice via inhibiting TGF-β1-Smad and non-Smad signaling pathway in vivo. Protein levels of p-Smad2, Smad2, p-Smad3, Smad3, p-P38, P38, p-JNK, JNK, p-ERK, ERK, p-AKT, and AKT were verified by Western blot in lung tissues. GAPDH was used as an internal reference in densitometric analysis. Data was presented as the means ± SD, n = 3. ** p < 0.01, *** p < 0.001.

6). Guizhi Fuling pill attenuates liver fibrosis in vitro and in vivo via inhibiting TGF-β1/Smad2/3 and activating IFN-γ/Smad7 signaling pathways. Bioengineered, 2022 (PubMed: 35387552) [IF=4.9]

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

Figure 2.| GZFL inhibits acetaldehyde-induced LX-2 cells activation through suppressing TGF-β1/Smad2/3 signaling and activating IFN-γ/STAT1/Smad7 signaling. LX-2 cells were exposed to acetaldehyde (AA; 400 μM) for 24 h, followed by exposure to colchicine (4 μg/ml) or GZFL (8 or 10 mg/ml) for another 24 h. (a, b) TGF-β1, TGF-βR2, CUGBP1, p-STAT1, p-Smad2, p-Smad3, Smad7,α-SMA and Collagen I expressions in LX-2 cells were detected by western blot assay.

7). Potential Effect of Non-Thermal Plasma for the Inhibition of Scar Formation: A Preliminary Report. Scientific Reports, 2020 (PubMed: 31974451) [IF=4.6]

Application: IHC    Species: rat    Sample:

Figure 5.|Non-thermal plasma down-regulated the expression of p-Smad 2/3. **p < 0.01. (A) Typical images of p-Smad 2 and p-Smad 3 afer immunohistochemical staining in the control and NTP groups. Te image in the lower lef corner is a magnifed view of the blue square. Bar = 200 μm/20 μm.

8). Calcium-sensing receptor activation attenuates collagen expression in renal proximal tubular epithelial cells. AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY, 2019 (PubMed: 30838870) [IF=4.2]

Application: WB    Species: human    Sample: primary human renal PTECs

Fig. 7. |Smad2 phosphorylation in primary human renal PTECs. (A-B) PTECs were incubated for 48 h with increasing concentrations of TGF-β1.

9). 2-Hydroxypropyl-β-cyclodextrin Regulates the Epithelial to Mesenchymal Transition in Breast Cancer Cells by Modulating Cholesterol Homeostasis and Endoplasmic Reticulum Stress. Metabolites, 2021 (PubMed: 34436503) [IF=4.1]

Application: WB    Species: Human    Sample: MDA-MB-231 cells

Figure 5 The effect of low concentrations of HP-β-CD on ER stress, TGF-β/Smad signaling pathway, and the distribution of cholesterol in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with various lower concentration of HP-β-CD (0, 0.2, 0.4, 0.6, 0.8 mmol/L). Expression levels of E-cadherin and Vimentin. (B) Expression levels of GRP78 and CHOP. (C) Expression level of p-Smad2 and Smad2. (D) MDA-MB-231 cells were treated with various lower concentration of HP-β-CD (0, 0.6, 0.8 mmol/L). Expression levels of HMGCR and LXRα. (E) Co-staining of Filipin and immunofluorescence analyses for the content of cholesterol in ER. Magnification, ×400. The red fluorescence showed the ER. Scale bars represent 40μm. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01 versus the control group.

10). Cyclin-dependent kinase subunit2 (CKS2) promotes malignant phenotypes and epithelial-mesenchymal transition-like process in glioma by activating TGFβ/SMAD signaling. Cancer Medicine, 2023 (PubMed: 36284444) [IF=4.0]

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