Phospho-Smad3 (Ser423+Ser425) Antibody

Product:	Phospho-Smad3 (Ser423+Ser425) Antibody
Catalog:	AF8315
Description:	Rabbit polyclonal antibody to Phospho-Smad3 (Ser423+Ser425)
Application:	WB IHC IF/ICC
Reactivity:	Human, Mouse, Rat
Prediction:	Pig, Bovine, Horse, Sheep, Rabbit, Chicken, Xenopus
Mol.Wt.:	58kDa; 48kD(Calculated).
Uniprot:	P84022
RRID:	AB_2840377

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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.

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MS Report

HPLC Report

MSDS

Data Sheet

COA

Protocols

WB Handbook

IHC Protocol

IF/ICC Protocol

Product Info

Source:

Rabbit

Application:

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.
*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%), Chicken(100%), Xenopus(100%)

Clonality:

Polyclonal

Specificity:

Phospho-Smad3 (Ser423+Ser425) Antibody detects endogenous levels of Smad3 only when phosphorylated at Ser423+Ser425.

RRID:

AB_2840377
Cite Format: Affinity Biosciences Cat# AF8315, RRID:AB_2840377.

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:

Uniprot:

P84022(SMAD3_HUMAN) >>Visit HPA database.

Gene(ID):

SMAD3(4088)

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

Xenopus

100

Chicken

100

Rabbit

100

Dog

Zebrafish

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

PTMs - P84022 As Substrate

P84022

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

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).

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.

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). Ganoderma lucidum polysaccharide inhibits HSC activation and liver fibrosis via targeting inflammation, apoptosis, cell cycle, and ECM-receptor interaction mediated by TGF-β/Smad signaling. Phytomedicine, 2023 (PubMed: 36603342) [IF=7.9]

2). 20(S)-ginsenoside Rg3 exerts anti-fibrotic effect after myocardial infarction by alleviation of fibroblasts proliferation and collagen deposition through TGFBR1 signaling pathways. Journal of Ginseng Research, 2023 [IF=6.3]

Application: WB Species: Mouse Sample: CFs

Fig. 6. TGFBR1 overexpression partly abolishes Rg3's inhibition on CFs growth, collagen synthesis, together with Smads activation. (A) CFs were infected with recombinant adenovirus for 48 h, and later stimulated by TGF-β1 (10 ng/ml) and Rg3 (20 μM), and Edu assay was performed to detect CFs proliferation (magnification, 200 × ). Red and blue fluorescence indicate proliferating cells as well as nuclei, separately. (B) Edu-positive cell proportion. (C) Expression of proliferation and collagen-related proteins in CFs following Ad-TGFBR1 or control adenovirus transfection. (D) Protein expression of TGFBR1 signaling in CFs after transfection. Relative PCNA (E), CDK6 (F), Cyclin D1 (G), collagen I (H), collagen III (I), p-TGFBR1 (J), p-Smad2 (K), and p-Smad3 (L) expression. Data are represented by mean ± SD for at least 3 groups. ∗p < 0.05, ∗∗p < 0.01. n.s, not significant.

3). Regorafenib-Attenuated, Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway. International Journal of Molecular Sciences, 2021 (PubMed: 33671452) [IF=5.6]

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.

4). Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis. Journal of Cellular and Molecular Medicine, 2021 (PubMed: 34750987) [IF=5.3]

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

5). Riboflavin protects against pancreatic cancer metastasis by targeting TGF-β receptor 1. Bioorganic chemistry, 2024 (PubMed: 38503026) [IF=5.1]

6). TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial-mesenchymal transition in diabetic nephropathy. Journal of Biological Chemistry, 2022 (PubMed: 35157852) [IF=4.8]

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.

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: 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.

8). Inhibition of p38 mitogen-activated protein kinases may attenuate scar proliferation after cleft lip surgery in rabbits via Smads signaling pathway. European Journal of Medical Research, 2022 (PubMed: 35858881) [IF=4.2]

Application: WB Species: rabbit Sample: fibroblast

Fig. 6 Effects of p38MAPK knockdown on extracellular matrix and Smads signaling pathway in fibroblast. A Effect of p38MAPK on the proliferation of scar fibroblasts was examined using the EdU assay. B mRNA expression levels of col I, col III, and α-SMA following p38MAPK knockdown as detected by RT-PCR. C Changes in protein expression of smad2, p-smad2, smad3, p-smad3, and α-SMA following p38MAPK knockdown as detected via western blotting. D Changes in protein expression of p-smad3 following p38MAPK knockdown as detected via immunofluorescence assay. Results are presented as the mean ± S.D,

9). Microvesicles-delivering Smad7 have advantages over microvesicles in suppressing fibroblast differentiation in a model of Peyronie's disease. BMC biotechnology, 2024 (PubMed: 38849776) [IF=3.5]

10). Liposomal Silybin Improves Glucose and Lipid Metabolisms in Type 2 Diabetes Mellitus Complicated with Non-Alcoholic Fatty Liver Disease via AMPK/TGF-β1/Smad Signaling. The Tohoku journal of experimental medicine, 2023 (PubMed: 37344419) [IF=2.2]

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Phospho-Smad3 (Ser423+Ser425) Antibody - #AF8315