Product: Phospho-Smad3 (Ser425) Antibody
Catalog: AF3362
Description: Rabbit polyclonal antibody to Phospho-Smad3 (Ser425)
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep, Rabbit, Dog, Chicken, Xenopus
Mol.Wt.: 58kDa; 48kD(Calculated).
Uniprot: P84022
RRID: AB_2834777

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Product Info

WB 1:500-1:2000, IHC 1:50-1:200, 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.

Pig(100%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(100%), Xenopus(100%)
Phospho-Smad3 (Ser425) Antibody detects endogenous levels of Smad3 only when phosphorylated at Serine 425.
Cite Format: Affinity Biosciences Cat# AF3362, RRID:AB_2834777.
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.


DKFZP586N0721; DKFZp686J10186; hMAD 3; hMAD-3; hSMAD3; HSPC193; HST17436; JV15 2; JV15-2; JV152; LDS1C; LDS3; MAD (mothers against decapentaplegic Drosophila) homolog 3; MAD homolog 3; Mad homolog JV15 2; Mad protein homolog; MAD, mothers against decapentaplegic homolog 3; Mad3; MADH 3; MADH3; MGC60396; Mothers against decapentaplegic homolog 3; Mothers against DPP homolog 3; SMA and MAD related protein 3; SMAD 3; SMAD; SMAD family member 3; SMAD, mothers against DPP homolog 3; Smad3; SMAD3_HUMAN;


Smad3 transcription factor phosphorylated and activated by TGF-beta-type receptors. A receptor-regulated Smad (R-smad). Binds directly to consensus DNA-binding elements in the promoters of target genes. In mouse required for establishemnt of the mucosal immune response and proper development of skeleton.



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 - P84022 As Substrate

Site PTM Type Enzyme
S2 Acetylation
S3 Phosphorylation
T8 Phosphorylation P11802 (CDK4) , P24941 (CDK2)
K13 Ubiquitination
K19 Acetylation
K29 Acetylation
K33 Ubiquitination
K36 Sumoylation
S37 Phosphorylation
K63 Ubiquitination
T66 Phosphorylation P49841 (GSK3B)
S78 Phosphorylation
K81 Ubiquitination
Y88 Phosphorylation
Y125 Phosphorylation
T132 Phosphorylation
T179 Phosphorylation P49336 (CDK8) , P28482 (MAPK1) , P24941 (CDK2) , P50750 (CDK9) , P11802 (CDK4) , P31749 (AKT1)
S204 Phosphorylation P28482 (MAPK1) , Q14680 (MELK) , Q16539 (MAPK14) , P27361 (MAPK3) , P11802 (CDK4)
S208 Phosphorylation P49336 (CDK8) , P11802 (CDK4) , P28482 (MAPK1) , Q16539 (MAPK14) , P50750 (CDK9)
S213 Phosphorylation P50750 (CDK9) , P49336 (CDK8) , P11802 (CDK4) , P28482 (MAPK1) , P24941 (CDK2)
S275 Phosphorylation
S309 Phosphorylation
K378 Acetylation
T388 Phosphorylation
T412 Phosphorylation
S416 Phosphorylation
S418 Phosphorylation P78368 (CSNK1G2)
S422 Phosphorylation P36897 (TGFBR1)
S423 Phosphorylation P36897 (TGFBR1)
S425 Phosphorylation P36897 (TGFBR1)

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 SMAD3/SMAD4 complex, activates transcription. Also can form a SMAD3/SMAD4/JUN/FOS complex at the AP-1/SMAD site to regulate TGF-beta-mediated transcription. Has an inhibitory effect on wound healing probably by modulating both growth and migration of primary keratinocytes and by altering the TGF-mediated chemotaxis of monocytes. This effect on wound healing appears to be hormone-sensitive. Regulator of chondrogenesis and osteogenesis and inhibits early healing of bone fractures. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.


Phosphorylated on serine and threonine residues. Enhanced phosphorylation in the linker region on Thr-179, Ser-204 and Ser-208 on EGF and TGF-beta treatment. Ser-208 is the main site of MAPK-mediated phosphorylation. CDK-mediated phosphorylation occurs in a cell-cycle dependent manner and inhibits both the transcriptional activity and antiproliferative functions of SMAD3. This phosphorylation is inhibited by flavopiridol. Maximum phosphorylation at the G(1)/S junction. Also phosphorylated on serine residues in the C-terminal SXS motif by TGFBR1 and ACVR1. TGFBR1-mediated phosphorylation at these C-terminal sites is required for interaction with SMAD4, nuclear location and transactivational activity, and appears to be a prerequisite for the TGF-beta mediated phosphorylation in the linker region. Dephosphorylated in the C-terminal SXS motif by PPM1A. This dephosphorylation disrupts the interaction with SMAD4, promotes nuclear export and terminates TGF-beta-mediated signaling. Phosphorylation at Ser-418 by CSNK1G2/CK1 promotes ligand-dependent ubiquitination and subsequent proteasome degradation, thus inhibiting SMAD3-mediated TGF-beta responses. Phosphorylated by PDPK1.

Acetylation in the nucleus by EP300 in the MH2 domain regulates positively its transcriptional activity and is enhanced by TGF-beta.

Poly-ADP-ribosylated by PARP1 and PARP2. ADP-ribosylation negatively regulates SMAD3 transcriptional responses during the course of TGF-beta signaling.

Ubiquitinated. Monoubiquitinated, leading to prevent DNA-binding. Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes. Ubiquitinated by RNF111, leading to its degradation: only SMAD3 proteins that are 'in use' are targeted by RNF111, RNF111 playing a key role in activating SMAD3 and regulating its turnover (By similarity). Undergoes STUB1-mediated ubiquitination and degradation.

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:15799969). Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1 (PubMed:16751101, PubMed:19289081). Co-localizes with LEMD3 at the nucleus inner membrane (PubMed:15601644). MAPK-mediated phosphorylation appears to have no effect on nuclear import (PubMed:19218245). PDPK1 prevents its nuclear translocation in response to TGF-beta (PubMed:17327236).

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

Monomer; in the absence of TGF-beta. Homooligomer; in the presence of TGF-beta. Heterotrimer; forms a heterotrimer in the presence of TGF-beta consisting of two molecules of C-terminally phosphorylated SMAD2 or SMAD3 and one of SMAD4 to form the transcriptionally active SMAD2/SMAD3-SMAD4 complex. Interacts with TGFBR1. Part of a complex consisting of AIP1, ACVR2A, ACVR1B and SMAD3. Interacts with AIP1, TGFB1I1, TTRAP, FOXL2, PML, PRDM16, HGS, WWP1 and SNW1. Interacts (via MH2 domain) with CITED2 (via C-terminus). Interacts with NEDD4L; the interaction requires TGF-beta stimulation. Interacts (via MH2 domain) with ZFYVE9. Interacts with HDAC1, TGIF and TGIF2, RUNX3, CREBBP, SKOR1, SKOR2, SNON, ATF2, SMURF2 and TGFB1I1. Interacts with DACH1; the interaction inhibits the TGF-beta signaling. Forms a complex with SMAD2 and TRIM33 upon addition of TGF-beta. Found in a complex with SMAD3, RAN and XPO4. Interacts in the complex directly with XPO4. Interacts (via MH2 domain) with LEMD3; the interaction represses SMAD3 transcriptional activity through preventing the formation of the heteromeric complex with SMAD4 and translocation to the nucleus. Interacts with RBPMS. Interacts (via MH2 domain) with MECOM. Interacts with WWTR1 (via its coiled-coil domain). Interacts (via the linker region) with EP300 (C-terminal); the interaction promotes SMAD3 acetylation and is enhanced by TGF-beta phosphorylation in the C-terminal of SMAD3. This interaction can be blocked by competitive binding of adenovirus oncoprotein E1A to the same C-terminal site on EP300, which then results in partially inhibited SMAD3/SMAD4 transcriptional activity. Interacts with SKI; the interaction represses SMAD3 transcriptional activity. Component of the multimeric complex SMAD3/SMAD4/JUN/FOS which forms at the AP1 promoter site; required for synergistic transcriptional activity in response to TGF-beta. Interacts (via an N-terminal domain) with JUN (via its basic DNA binding and leucine zipper domains); this interaction is essential for DNA binding and cooperative transcriptional activity in response to TGF-beta. Interacts with PPM1A; the interaction dephosphorylates SMAD3 in the C-terminal SXS motif leading to disruption of the SMAD2/3-SMAD4 complex, nuclear export and termination of TGF-beta signaling. 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 MEN1. Interacts with IL1F7. Interaction with CSNK1G2. Interacts with PDPK1 (via PH domain). Interacts with DAB2; the interactions are enhanced upon TGF-beta stimulation. Interacts with USP15. Interacts with PPP5C; the interaction decreases SMAD3 phosphorylation and protein levels. Interacts with LDLRAD4 (via the SMAD interaction motif). Interacts with PMEPA1. Interacts with ZC3H3 (By similarity). Interacts with ZNF451. Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4. Interacts with ZFHX3. Interacts weakly with ZNF8. Interacts (when phosphorylated) with RNF111; RNF111 acts as an enhancer of the transcriptional responses by mediating ubiquitination and degradation of SMAD3 inhibitors (By similarity). Interacts with STUB1, HSPA1A, HSPA1B, HSP90AA1 and HSP90AB1. Interacts (via MH2 domain) with ZMIZ1 (via SP-RING-type domain); in the TGF-beta signaling pathway increases the activity of the SMAD3/SMAD4 transcriptional complex.


The MH1 domain is required for DNA binding. Also binds zinc ions which are necessary for the DNA binding.

The MH2 domain is required for both homomeric and heteromeric interactions and for transcriptional regulation. Sufficient for nuclear import.

The linker region is required for the TGFbeta-mediated transcriptional activity and acts synergistically with the MH2 domain.

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 > Wnt 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 > Hepatitis B.

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

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

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

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

· Human Diseases > Cancers: Specific types > Chronic myeloid leukemia.   (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). Roscovitine attenuates renal interstitial fibrosis in diabetic mice through the TGF-β1/p38 MAPK pathway. BIOMEDICINE & PHARMACOTHERAPY, 2019 (PubMed: 31029000) [IF=7.5]

Application: WB    Species: human    Sample: HK2 cells

Fig. 5. |Effect of roscovitine on the expression of α-SMA, E-Cadherin, collagen I, TGF-β1, p-Smad3 and p-p38 MAPK in high glucose treated HK2. (A) Representative western blot showing α-SMA, E-Cadherin and collagen I. (B) Representative western blot showing TGF-β1, p-Smad3 and p-p38 MAPK. (C)-(H) Quantitative analyses of the results from western blot. The each protein level was normalized to β-actin. *P < 0.05, n=3.

2). Timosaponin BII inhibits TGF‐β mediated epithelial‐mesenchymal transition through Smad‐dependent pathway during pulmonary fibrosis. PHYTOTHERAPY RESEARCH, 2023 (PubMed: 36807664) [IF=7.2]

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 6 Remdesivir inhibits TGF-β1-induced activation of Smad and non-Smad signaling pathway in lung fibroblasts (A) Luciferase assays of CAGA-NIH3T3 cells. Cells were pretreated with Remdesivir (0–50 μM) for 30 min and then incubated with TGF-β1 (5 ng ml−1) for 24 h, then analyzed with luciferase assay. SB431542 is a TGF-β1/Smad pathway inhibitor and serves as a positive control (B) NIH-3T3 cells were co-treated with TGF-β1 (5 ng ml−1) and Remdesivir (12.5, 25, 50 μM) for 1 h. P-Smad3 and Smad3 were assessed using western blot. GAPDH was used as the internal control (C) PPF cells were co-treated with TGF-β1 (5 ng ml−1) and Remdesivir (12.5, 25, 50 μM) for 1 h. P-Smad3 and Smad3 were assessed using western blot. GAPDH was used as the internal control (D) NIH-3T3 cells were co-treated with TGF-β1 (5 ng ml−1) and Remdesivir (12.5, 25, 50 μM) for 1 h and the phosphorylation levels of P-38, JNK, ERK and Akt were analyzed by Western blot. β-tubulin was used as a loading control in grayscale analysis. Scale bar = 60 μm. Data was presented as the means ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

6). Ribes diacanthum Pall (RDP) ameliorates UUO-induced renal fibrosis via both canonical and non-canonical TGF-β signaling pathways in mice. JOURNAL OF ETHNOPHARMACOLOGY, 2019 (PubMed: 30342194) [IF=5.4]

Application: WB    Species: mouse    Sample: renal

Figure 5.| RDP treatment regulates UUO-induced TGF-β1/Smads signaling pathway in UUO-induced renal fibrosis mice. Representative western blots and quantification of the levels of total and phosphorylated Smad2 (A) and Smad3 (B) as well as total Smad7 (C) in sham, RDP, UUO and RDP+UUO groups. Data are presented as mean ±SEM (n = 3 in each group). #P < 0.05, ##P < 0.01, UUO group vs. sham group; *P <0.05, **P < 0.01, UUO + RDP group vs. UUO group.

7). miR‑29b suppresses proliferation and induces apoptosis of hepatocellular carcinoma ascites H22 cells via regulating TGF‑β1 and p53 signaling pathway. INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 2021 (PubMed: 34184070) [IF=5.4]

Application: WB    Species: Mice    Sample: H22 cells

Figure 4 Effect of miR-29b on the TGF-β1 signaling pathway in H22 cells. (A and B) Western blot analysis of TGF-β1, p-Smad3 and Smad7. (C-E) Protein intensities of TGF-β1, p-Smad3 and Smad7. Values represent the means ± SD (n=3). (F-H) mRNA levels of TGF-β1, Smad3 and Smad7. Values represent the means ± SD (n=5). #P<0.05 and ##P<0.01 vs. control mimic; *P<0.05 and **P<0.01 vs. control inhibitor. miR, microRNA; TGF, transforming growth factor; SD, standard deviation; p-, phosphorylated.

8). Exosome‑encapsulated miR‑26a attenuates aldosterone‑induced tubulointerstitial fibrosis by inhibiting the CTGF/SMAD3 signaling pathway. International Journal of Molecular Medicine, 2023 (PubMed: 36524378) [IF=5.4]

Application: WB    Species: Mice    Sample: kidneys

Figure 6 miR-26a/CTGF inhibits SMAD3 activation. (A) Western blot analysis of SMAD3 and p-SMAD3 protein expression levels in the kidneys of mice in the sham, ALD, ALD + Exo-NC and ALD + Exo-miR-26a groups. (B) Western blot analysis of SMAD3 and p-SMAD3 protein expression levels in mTECs co-transfected with oe-CTGF or oe-NC and miR-26a mimic or NC mimic for 6 h, and then treated with ALD (1×10−6 M) for 48 h. (C) Western blot analysis of SMAD3 and p-SMAD3 protein levels in mTECs co-transfected with si-CTGF or si-NC and miR-26a inhibitor or NC inhibitor for 6 h, and then treated with ALD (1×10−6 M) for 48 h. Data are presented as mean ± SD; Data are presented as the mean ± SD; *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ##P<0.01. ALD, aldosterone; CTGF, connective tissue growth factor; Exo, exosome encapsulated; miR, microRNA; mTEC, mouse tubular epithelial cells; NC, negative control; p-phosphorylated.

9). Mesenchymal stem cells ameliorate silica‐induced pulmonary fibrosis by inhibition of inflammation and epithelial‐mesenchymal transition. JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, 2021 (PubMed: 34076355) [IF=5.3]

Application: WB    Species: rat    Sample: lung

FIGURE 5|BMSCs blocked the activation of TGF-β/Smad pathway. (D) Western blot results of TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3 and Smad7 protein expression levels. n = 3 rats per group.

10). 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.

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