Product: Phospho-Smad2 (Ser465+Ser467) Antibody
Catalog: AF8314
Source: Rabbit
Application: WB, IHC, IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Zebrafish, Bovine, Sheep, Rabbit, Dog, Xenopus
Mol.Wt.: 58kDa; 52kD(Calculated).
Uniprot: Q15796
RRID: AB_2840376

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Product Info

WB 1:1000-3000, IF/ICC 1:100-1:500, IHC 1:50-1:200
*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.

Pig(100%), Zebrafish(100%), Bovine(100%), Sheep(100%), Rabbit(100%), Dog(100%), Xenopus(100%)
Phospho-Smad2 (Ser465+Ser467) Antibody detects endogenous levels of Smad2 only when phosphorylated at Ser465+Ser467.
Cite Format: Affinity Biosciences Cat# AF8314, RRID:AB_2840376.
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.




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). Sun X et al. ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-β receptor II in breast cancer. J Exp Clin Cancer Res 2021 Apr 30;40(1):149. (PubMed: 33931075) [IF=7.068]

Application: IF/ICC    Species: mouse    Sample: ALG3-sg cells

Fig. 5| ALG3 enhances radioresistance via regulation of TGFBR2 glycosylation. (a) Downshift of TGFBR2 bands in ALG3-sg cells was detected by Western blot. But not TGFBR1 bands (b) Representative immunofluorescence images of TGFBR2 expression level in cytoplasmic and membrane fractions. (e) Representative immunofluorescence images of p-SMAD2 expression level in cytoplasmic and nuclear fractions. Nuclear translocation of p-SMAD2 was significantly decreased in ALG3-sg and tunicamycin treatment groups.

2). Dai Z et al. The critical role of B4GALT4 in promoting microtubule spindle assembly in HCC through the regulation of PLK1 and RHAMM expression. J Cell Physiol 2022 Jan;237(1):617-636. (PubMed: 34270095) [IF=6.513]

Application: WB    Species: Human    Sample: HepG2 cells

FIGURE 9 B4GALT4 knockdown regulated TGF‐beta1/TGF‐beta receptor‐1 signaling pathway and modified the expressions of PLK1 and RHAMM. (a) B4GALT4 knockdown downregulated the expression of lumican. (b) The expressions of B4GALT4, P‐Smad2, Smad2, PLK1, and RHAMM in HepG2 cells after different treatments as mentioned above were detected by Western blot analysis. (c) The comparison of the mRNA expression level of HMMR between tumor and normal samples. (d) Survival analysis of HMMR in HCC. (e) The protein interaction network diagram of RHAMM, PLK1, and other pathway‐related genes. (f) ChIP‐seq results showed that Smad2/3 and p300 binding regions are enriched around the promoter of PLK1. (g) Schematic illustration of the B4GALT4‐mediated regulatory network responsible for the microtubule spindle assembly in HCC (****p < 0.0001). B4GALT, beta 1,4‐galactosyltransferase; HCC, hepatocellular carcinoma; mRNA, messenger RNA

3). Liu T et al. Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis. J Cell Mol Med 2021 Nov 9. (PubMed: 34750987) [IF=5.295]

Application: WB    Species: Human    Sample: LX‐2 cells

FIGURE 4 Liver fibrosis‐related protein expressions under Smad2/3, PI3K/Akt inhibitors in LX‐2 cells (A‐D) Western blot analysis showed the protein expressions of α‐SMA, collagen I, p‐Smad2, p‐Smad3, Samd2/3, p‐PI3K, PI3K, p‐Akt and Akt in control, SIS3/LY294002, sTREM‐1 and sTREM‐1+SIS3/LY294002 groups of LX‐2 cells. ns: not significant, * p < 0.05, ** p < 0.01

4). Wang XF et al. Potential Effect of Non-Thermal Plasma for the Inhibition of Scar Formation: A Preliminary Report. Sci Rep 2020 Jan 23;10(1):1064. (PubMed: 31974451) [IF=4.996]

Application: WB    Species: Rat    Sample: scar tissue

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 after immunohistochemical staining in the control and NTP groups. The image in the lower left corner is a magnified view of the blue square. Bar = 200 μm/20 μm. (B) Statistical analysis of p-Smad2/3 on day 21 in the control and the NTP treated wounds.

5). Liu S et al. Sphingomyelin synthase 1 regulates the epithelial‑to‑mesenchymal transition mediated by the TGF‑β/Smad pathway in MDA‑MB‑231 cells. Mol Med Rep 2018 Dec 4 (PubMed: 30535436) [IF=3.423]

Application: WB    Species: human    Sample: MDA?MB?231 cells

Figure 4.| Overexpression of SMS1 regulates TGF?β1?induced EMT via TGF?β type I receptors. (A)?Expression of TβRI and (B)?phosphorylation of Smad2 and Smad2 were measured by western blotting in MDA?MB?231 cells.

6). Wu Z et al. Jinlian Xiaodu Decoction Protects against Bleomycin-Induced Pulmonary Fibrosis in Rats. Evid Based Complement Alternat Med 2022 Jun 23;2022:4206364. (PubMed: 35783517) [IF=2.650]

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