Phospho-Smad3 (Ser425) Antibody | Affinity Biosciences

Product:	Phospho-Smad3 (Ser425) Antibody
Catalog:	AF3362
Description:	Rabbit polyclonal antibody to Phospho-Smad3 (Ser425)
Application:	WB IHC IF/ICC
Cited expt.:	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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Blocking peptides are peptides that bind specifically to the target antibody and block antibody binding. These peptide usually contains the epitope recognized by the antibody. Antibodies bound to the blocking peptide no longer bind to the epitope on the target protein. By comparing the staining from the blocked antibody versus the antibody alone, one can see which staining is specific.

Size	Price	Inventory
100ul	$280	In stock
200ul	$350	In stock

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MSDS

Data Sheet

COA

Protocols

WB Handbook

IHC Protocol

IF/ICC Protocol

Product Info

Source:

Rabbit

Application:

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.
*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%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(100%), Xenopus(100%)

Clonality:

Polyclonal

Specificity:

Phospho-Smad3 (Ser425) Antibody detects endogenous levels of Smad3 only when phosphorylated at Serine 425.

RRID:

AB_2834777
Cite Format: Affinity Biosciences Cat# AF3362, RRID:AB_2834777.

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

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;

Immunogens

Immunogen:

A synthesized peptide derived from human Smad3 around the phosphorylation site of Ser425.

Uniprot:

P84022(SMAD3_HUMAN) >>Visit HPA database.

Gene(ID):

SMAD3(4088)

Description:

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.

Sequence:

P84022 SMAD3_HUMAN:
Protein BLAST With
NCBI/
ExPASy/
Uniprot

MSSILPFTPPIVKRLLGWKKGEQNGQEEKWCEKAVKSLVKKLKKTGQLDELEKAITTQNVNTKCITIPRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELRAMELCEFAFNMKKDEVCVNPYHYQRVETPVLPPVLVPRHTEIPAEFPPLDDYSHSIPENTNFPAGIEPQSNIPETPPPGYLSEDGETSDHQMNHSMDAGSPNLSPNPMSPAHNNLDLQPVTYCEPAFWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS

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

Horse

100

Bovine

100

Sheep

100

Dog

100

Xenopus

100

Chicken

100

Rabbit

100

Zebrafish

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

Research Backgrounds

P84022_SMAD3_HUMAN

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 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.

PTMs:

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).

Family&Domains:

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.

References

1). Roscovitine attenuates renal interstitial fibrosis in diabetic mice through the TGF-β1/p38 MAPK pathway. BIOMEDICINE & PHARMACOTHERAPY, 2019 (PubMed: 31029000) [IF=6.9]

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). L-Glutamine alleviates osteoarthritis by regulating lncRNA-NKILA expression through the TGF-β1/SMAD2/3 signalling pathway. Clinical Science, 2022 (PubMed: 35730575) [IF=6.7]

3). Timosaponin BII inhibits TGF‐β mediated epithelial‐mesenchymal transition through Smad‐dependent pathway during pulmonary fibrosis. PHYTOTHERAPY RESEARCH, 2023 (PubMed: 36807664) [IF=6.1]

4). MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4. iScience, 2024 (PubMed: 38974966) [IF=5.8]

5). 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=5.7]

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.

6). 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=5.7]

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.

7). 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.

8). Myricetin ameliorates bleomycin-induced pulmonary fibrosis in mice by inhibiting TGF-β signaling via targeting HSP90β. BIOCHEMICAL PHARMACOLOGY, 2020 (PubMed: 32535102) [IF=5.3]

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). IL-22 inhibits bleomycin-induced pulmonary fibrosis in association with inhibition of IL-17A in mice. Arthritis Research & Therapy, 2022 (PubMed: 36564791) [IF=4.9]

Application: WB Species: Mouse Sample: A549 cells, NIH/3T3 cells

Fig. 3 IL-22 inhibits collagen production of A549 cells, NIH/3T3 cells, and MLFs in vitro. A A549 cells and NIH/3T3 cells were cultured with 5 ng/ml TGF-β1 with or without IL-22 (1 ng/ml) for 48 h. MLFs were cultured with 5 ng/ml TGF-β1 with or without IL-22 (1 ng/ml, 5 ng/ml) for 48 h. Western blotting was used to detect proteins (n = 3). Relative intensity of each band was normalized to GAPDH protein. The relevant gels and blots were cropped. (B) MLFs were cultured with 5 ng/ml TGF-β1 with or without IL-22(1 ng/ml, 5 ng/ml, 10 ng/ml) for 48 h. Western blotting was used to detect proteins as indicated (n = 3). Relative intensity of each band was normalized to GAPDH protein. The relevant gels and blots were cropped. C A549 cells, NIH/3T3 cells, and MLFs were cultured with gradient doses of IL-22 for 24 h, 48 h, and 72 h in the presence of 5 ng/ml TGF-β1. Cell viability was measured using the CCK-8 assay (n = 6). Viability of cells without IL-22 treatment was set as 100%. D NIH/3T3 cells without TGF-β1 were co-cultured with T cells of WT or IL-22KO mice for 24 h with or without IL-22(10 ng/ml), and T cells were activated with CD3 and CD28. Relative intensity of each band was normalized to GAPDH protein. The relevant gels and blots were cropped. Data are mean ± SEM, compared using one-way ANOVA test.

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Phospho-Smad3 (Ser425) Antibody - #AF3362