Phospho-Smad4 (Thr276)[Thr277] Antibody - #AF8316

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Product: Phospho-Smad4 (Thr276)[Thr277] Antibody
Catalog: AF8316
Description: Rabbit polyclonal antibody to Phospho-Smad4 (Thr276)[Thr277]
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep, Rabbit, Dog
Mol.Wt.: 70kDa; 60kD(Calculated).
Uniprot: Q13485
RRID: AB_2840378

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Related Downloads
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, 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(90%), Bovine(90%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(90%)
Clonality:
Polyclonal
Specificity:
Phospho-Smad4 (Thr276) Antibody detects endogenous levels of Smad4 only when phosphorylated at Thr277, which site historically referenced as Thr276.
RRID:
AB_2840378
Cite Format: Affinity Biosciences Cat# AF8316, RRID:AB_2840378.
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

(Small) Mothers Against Decapentaplegic; Deleted in Pancreatic Carcinoma 4; Deleted in Pancreatic Carcinoma; Deleted in pancreatic carcinoma locus 4; Deletion target in pancreatic carcinoma 4; DPC 4; DPC4; hSMAD4; JIP; MAD homolog 4; MAD mothers against decapentaplegic Drosophila homolog 4; MAD mothers against decapentaplegic homolog 4; MADH 4; MADH4; Med; Medea; Mothers against decapentaplegic homolog 4; Mothers against decapentaplegic, Drosophila, homolog of, 4; Mothers against DPP homolog 4; MYHRS; OTTHUMP00000163548; SMA- and MAD-related protein 4; SMAD 4; SMAD family member 4; SMAD mothers against DPP homolog 4; SMAD4; SMAD4_HUMAN;

Immunogens

Immunogen:
Uniprot:

Q13485(SMAD4_HUMAN) >>Visit HPA database.

Gene(ID):
SMAD4(4089)
Sequence:
MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITAITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFDLKCDSVCVNPYHYERVVSPGIDLSGLTLQSNAPSSMMVKDEYVHDFEGQPSLSTEGHSIQTIQHPPSNRASTETYSTPALLAPSESNATSTANFPNIPVASTSQPASILGGSHSEGLLQIASGPQPGQQQNGFTGQPATYHHNSTTTWTGSRTAPYTPNLPHHQNGHLQHHPPMPPHPGHYWPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD

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
Horse
100
Sheep
100
Rabbit
100
Pig
90
Bovine
90
Dog
90
Xenopus
0
Zebrafish
0
Chicken
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - Q13485 As Substrate

Site PTM Type Enzyme
T9 Phosphorylation Q9UBE8 (NLK)
S22 Phosphorylation
S32 Phosphorylation
K37 Acetylation
K45 Acetylation
K45 Ubiquitination
K48 Acetylation
K70 Ubiquitination
T77 Phosphorylation Q15831 (STK11)
Y95 Phosphorylation
K106 Acetylation
K113 Sumoylation
K113 Ubiquitination
K122 Ubiquitination
S138 Phosphorylation Q9UBE8 (NLK)
K159 Sumoylation
S178 Phosphorylation
T265 Phosphorylation
T269 Phosphorylation
T273 Phosphorylation
T277 Phosphorylation P28482 (MAPK1) , P27361 (MAPK3)
S343 Phosphorylation Q14680 (MELK)
K385 Ubiquitination
K428 Acetylation
S504 Phosphorylation
K507 Acetylation
K507 Ubiquitination
Y513 Phosphorylation
K519 Ubiquitination

Research Backgrounds

Function:

In muscle physiology, plays a central role in the balance between atrophy and hypertrophy. When recruited by MSTN, promotes atrophy response via phosphorylated SMAD2/4. MSTN decrease causes SMAD4 release and subsequent recruitment by the BMP pathway to promote hypertrophy via phosphorylated SMAD1/5/8. Acts synergistically with SMAD1 and YY1 in bone morphogenetic protein (BMP)-mediated cardiac-specific gene expression. Binds to SMAD binding elements (SBEs) (5'-GTCT/AGAC-3') within BMP response element (BMPRE) of cardiac activating regions (By similarity). Common SMAD (co-SMAD) is the coactivator and mediator of signal transduction by TGF-beta (transforming growth factor). Component of the heterotrimeric SMAD2/SMAD3-SMAD4 complex that forms in the nucleus and is required for the TGF-mediated signaling. Promotes binding of the SMAD2/SMAD4/FAST-1 complex to DNA and provides an activation function required for SMAD1 or SMAD2 to stimulate transcription. Component of the multimeric SMAD3/SMAD4/JUN/FOS complex which forms at the AP1 promoter site; required for synergistic transcriptional activity in response to TGF-beta. May act as a tumor suppressor. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.

PTMs:

Phosphorylated by PDPK1.

Monoubiquitinated on Lys-519 by E3 ubiquitin-protein ligase TRIM33. Monoubiquitination hampers its ability to form a stable complex with activated SMAD2/3 resulting in inhibition of TGF-beta/BMP signaling cascade. Deubiquitination by USP9X restores its competence to mediate TGF-beta signaling.

Subcellular Location:

Cytoplasm. Nucleus.
Note: Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with R-SMAD (PubMed:15799969). 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:

Found in a complex with SMAD1 and YY1 (By similarity). Interacts with CITED2 (By similarity). Monomer; in the absence of TGF-beta activation. Heterodimer; on TGF-beta activation. Composed of two molecules of a C-terminally phosphorylated R-SMAD molecule, SMAD2 or SMAD3, and one molecule of SMAD4 to form the transcriptional active SMAD2/SMAD3-SMAD4 complex. Found in a ternary complex composed of SMAD4, STK11/LKB1 and STK11IP. Interacts with ATF2, COPS5, DACH1, MSG1, SKI, STK11/LKB1, STK11IP and TRIM33. Interacts with ZNF423; the interaction takes place in response to BMP2 leading to activation of transcription of BMP target genes. Interacts with ZNF521; the interaction takes place in response to BMP2 leading to activation of transcription of BMP target genes. Interacts with USP9X. Interacts (via the MH1 and MH2 domains) with RBPMS. Interacts with WWTR1 (via coiled-coil domain). 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 with CITED1. Interacts with PDPK1 (via PH domain) (By similarity). Interacts with VPS39; this interaction affects heterodimer formation with SMAD3, but not with SMAD2, and leads to inhibition of SMAD3-dependent transcription activation. Interactions with VPS39 and SMAD2 may be mutually exclusive. Interacts with ZC3H3 (By similarity). Interacts (via MH2 domain) with ZNF451 (via N-terminal zinc-finger domains). Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4. Interacts weakly with ZNF8. Interacts with NUP93 and IPO7; translocates SMAD4 to the nucleus through the NPC upon BMP7 stimulation resulting in activation of SMAD4 signaling. Interacts with CREB3L1, the interaction takes place upon TGFB1 induction and SMAD4 acts as CREB3L1 coactivator to induce the expression of genes involved in the assembly of collagen extracellular matrix. Interacts with DLX1. Interacts with ZBTB7A; the interaction is direct and stimulated by TGFB1. Interacts with CREBBP; the recruitment of this transcriptional coactivator is negatively regulated by ZBTB7A. Interacts with EP300; the interaction with this transcriptional coactivator is negatively regulated by ZBTB7A. Interacts with HDAC1. 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.

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

Belongs to the dwarfin/SMAD family.

Research Fields

· Cellular Processes > Cell growth and death > Cell cycle.   (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: 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)

· Organismal Systems > Immune system > Th17 cell differentiation.   (View pathway)

References

1). Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling. eBioMedicine, 2020 (PubMed: 32512510) [IF=11.1]

Application: WB    Species: Human    Sample: HCT116 cells

Fig. 5. DACH1 promotes adenomas organoid formation via modulating BMP signalling. a DACH1 overexpression induced upregulation of cancer stem cell marker genes. b and c. Gene Ontology (GO) analysis showed that DACH1 overexpression induced the upregulation of stem cell signature genes and the downregulation of cell cycle arrest signature genes. d and e. DACH1 induced the downregulation of ATOH8, TGFb3, MSX1, NBL1, BMP7, and FKBP4 and the upregulation of FKBP8, ID1, and MAPK3 (experiments were performed in triplicate). f. IF images showing the colocalization of SMAD4 and DACH1 in the nuclei; scale bars=20 mm g-h. DACH1 coprecipitated endogenous SMAD4. Reverse immunoprecipitation was confirmed with an antiSMAD4 antibody. i. DACH1 overexpression increased the protein level of SMAD4 and decreased the level of phosphorylated SMAD4 (Thr276) [Thr277]. j and k. DACH1 knockdown led to an increase of the mRNA levels of NBL1 and BMP7 compared with the shNC group, and siRNA mediated SMAD4 knockdown in HCT116-shDACH1 cells eliminated the increase. l. DACH1 overexpression was sufficient to compensate for the withdrawal of Noggin and supported the formation of adenoma organoids. m. Addition of Noggin into the culture medium for 24 and 36 h decreased the mRNA levels of NBL1 and BMP7, which were increased by DACH1 knockdown in HCT116 cells. n and o. Overexpression of DACH1 upregulates LGR5, Notch1 and the protein level of NICD, while did not induce significant upregulation of HES1. Scale bars=20 mm. For 5d, 5e, 5k, 5 m and 5n, **P < 0.01, *P < 0.05, Student’s t-test. Error bars: mean§SD.
2). SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP/TGF-β cell signaling pathway. Cell Death & Disease, 2019 (PubMed: 31699966) [IF=9.0]

Application: WB    Species: Mouse    Sample: C2C12 cells

Fig. 6 SPARCL1 regulates BMP7 expression and BMP/TGF-β cell signaling pathway. a, d shows the protein expression of BMP7 regulated by SPARCL1 activation and inhibition, respectively. C2C12 cells were induced to differentiate at 72 h. pSPgRNA-S-2 is the SPARCL1 activation group, while pSPgRNA is the blank control for SPARCL1 activation. NC is the negative control for SPARCL1 siRNA interference. b, c are greyscale scans of SPARCL1 and BMP7 proteins in A. e, f are grayscale scans of SPARCL1 and BMP7 proteins in D. i and p shows changes in the expression of BMP/ TGF-β-associated proteins when SPARCL1 was activated and inhibited, respectively, and C2C12 cells were induced to differentiate at 72 h. pSPgRNA-S-2 is the SPARCL1 activation group, while pSPgRNA is the blank control for SPARCL1 activation. NC is the negative control for SPARCL1 siRNA interference. j–o are greyscale scans of proteins in I. q–v are greyscale scans of proteins in P. **P values < 0.01 were considered as significant
3). Methylation of BRD4 by PRMT1 regulates BRD4 phosphorylation and promotes ovarian cancer invasion. Cell death & disease, 2023 (PubMed: 37737256) [IF=9.0]

Application: WB    Species: Mouse    Sample: A2780 cells

Fig. 7 PRMT1-mediated BRD4 methylation is correlated with TGF-β. a and b IB analysis of TGF-β derived from SKOV3 cells and A2780 cells transfected with shPRMT1. c and d IB analysis of TGF-β derived from SKOV3 cells and A2780 cells treated with AMI-1 for 24, 48, and 72 h. e–g IB analysis of TGF-β derived from SKOV3 cells, A2780 cells, 293T cells stably expressed HA-BRD4-WT and HA-BRD4-R3K mutant. h and i IB analysis of TGF-β derived from BRD4 knockdown 293T cells and BRD4 knockdown A2780 cells stably expressed HA-BRD4-WT and HA-BRD4-R3K mutant. j IB analysis of p-Smad2 and p-Smad4 derived from A2780 cells transfected with shPRMT1-1 and shPRMT1-2. k IB analysis of TGF-β derived from A2780 cells infected with sgTGF-β. l CCK8 assays were performed in TGF-β knockout A2780 ovarian cancer cells for 24, 48, and 72 h. Statistical analysis of relative colony numbers was plotted. ****P 
4). Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-β/Smad pathway in 3T6 and HFL-1 cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2022 (PubMed: 36068772) [IF=7.5]

Application: WB    Species: Human    Sample: fibroblasts

Fig. 7. The effects of DTF1 and DTF2 on regulating the TGF-β/Smad pathway. (A) Representative Western blotting images of TGF-β1, α-SMA, Smad3, and p-Smad3. The protein expression of (B) α-SMA, (C) TGF-β1, (E) Smad3, and (F) p-Smad3/Smad3 were determined. (D) Representative Western blotting images of Smad4, p-Smad4, and Smad7. The protein expression of (G) Smad4, (H) p-Smad4/Smad4, and (I) Smad7 were determined. ##P 
5). TOPK mediates immune evasion of renal cell carcinoma via upregulating the expression of PD-L1. iScience, 2022 (PubMed: 37404377) [IF=5.8]

Application: WB    Species: Human    Sample: 293T cells

Figure 6 TOPK promotes PD-L1 expression in RCC by enhancing TGF-β/Smad pathway activity The expression of different proteins in different cells was detected by immunoblot analysis. (A) Detection of TOPK binding to Smad4 by immunoprecipitation. (B-C) The expression of TOPK and Smad4 in TOPK knockdown 786-O cells(B) and overexpressed Caki-1 cells(C) were detected, respectively. (D) The expression of TOPK, p-TOPK(Thr9), Smad4, p-Smad4 (Thr276), Smad2/3, p-Smad2/3 (S465, 467/S423, 425), and PD-L1 was detected in 293T cells with or without TGF-β1 (5 ng/ml, 2h). (E-F) The expression of TOPK, p-TOPK(Thr9), Smad4, p-Smad4 (Thr276), Smad2/3, p-Smad2/3 (S465, 467/S423, 425), and PD-L1 was detected in TOPK knockdown (E) or overexpressed cells(F) with or without TGF-β1 (5 ng/ml, 2h). (G) Expression of TOPK, p-TOPK(Thr9), Smad4, p-Smad4 (Thr276), Smad2/3, p-Smad2/3 (S465, 467/S423, 425), and PD-L1 was detected in TOPK overexpressing cells alone or in combination with TGF-β1 (5 ng/mL, 2h) and OTS964 (10nM, 24h). See also Figure S4.
6). Does Chronic Pancreatitis in Growing Pigs Lead to Articular Cartilage Degradation and Alterations in Subchondral Bone?. International journal of molecular sciences, 2024 (PubMed: 38396667) [IF=5.6]

Application: IHC    Species: pig    Sample:

Figure 6 (a) Representative immunohistochemical (IHC) staining for TGF-β1, TGFβR1, TGFβR2, pSMAD2, and pSMAD4 in the articular cartilage of the distal femur from control (CONT) and chronic pancreatitis (CP) pigs. Scale bars represent 50 μm. (b) Bar graphs illustrating the optical density (OD) of expression for the analyzed proteins. Measurements were conducted on two histological samples per animal in at least ten randomly selected areas. Bars indicate mean values ± standard error (n = 5 pigs per group). Statistical significance is denoted as * p < 0.05; ** p < 0.01; *** p < 0.001.
7). MYOC Promotes the Differentiation of C2C12 Cells by Regulation of the TGF-β Signaling Pathways via CAV1. Geobiology, 2021 (PubMed: 34356541) [IF=3.7]

Application: WB    Species: Mice    Sample: C2C12 cell

Figure 4 Effects of CAV1 on C2C12 cell differentiation. (A). Change in CAV1, MYH2, and MYOG expression levels at different stages of C2C12 cell differentiation. (B). Grayscale scan of CAV1 in Figure A. (C). Grayscale scan of MYH2 in Figure A. (D). Grayscale scan of MYOG in Figure A. (E). Immunofluorescence was used to measure the changes in CAV1 expression levels and the morphology of myotubes at various stages of C2C12 differentiation. CAV1 was labeled green using FITC and the nucleus blue using DAPI. (F). Laser confocal microscopy demonstrating the location of CAV1 expression, stained red using RBFITC, and nuclei blue using DAPI. (G). Changes in CAV1, MYH2, and MYOG expression levels after siRNA transfection for 48 h and 72 h. (H). Grayscale scan of CAV1 in Figure G. (I). Grayscale scan of MYH2 in Figure G. (J). Grayscale scan of MYOG in Figure G. (K). Changes in the fusion rate of C2C12 cells in mice after CAV1 inhibition by immunofluorescence detection. (L). Myotubule fusion rate in Figure K. (M). Change in CAV1, P-Smad2, and P-Smad4 expression after siRNA fragment S1 transfection of CAV1. (N). Grayscale scan of CAV1 in Figure M. (O). Grayscale scan of P-Smad2 in Figure M. (P). Grayscale scan of P-Smad4 in Figure M. (** p values < 0.01, * p values < 0.05) (n = 3).
8). Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells. Biomedical reports, 2023 (PubMed: 37954636) [IF=2.3]

Application: WB    Species: Human    Sample: L929 fibroblasts

Figure 7 Effect of Oct-LMSG on Smad signaling protein expression in L929 fibroblasts. Smad2/3, Smad4, p-Smad2/3 and p-Smad4 protein levels were detected by western blot assay after treatment with either Oct-LMSG (100 µg/ml) or TGF-β (10 ng/ml) for 6, 12 and 24 h relative to β-actin. The values are expressed as the mean ± standard error of the mean of three independent experiments. *P

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