Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46] - BSA and Azide free
Catalog# HA752027
Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46] - BSA and Azide free
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WB
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IHC-P
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IF-Cell
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Human
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Mouse
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Rat
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unconjugated
Overview
Product Name
Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46] - BSA and Azide free
Antibody Type
Recombinant Rabbit monoclonal Antibody
Immunogen
Synthetic phosphopeptide corresponding to residues surrounding Ser463/465 of human SMAD1 and SMAD5 protein.
Product Specificity
Phospho-Smad1/5/9 (S463/S465/S467) (JE59-46) Rabbit mAb recognizes endogenous levels of SMAD1 and SMAD5 protein when phosphorylated at Ser463/465 and SMAD9 protein when phosphorylated at Ser465/467.
Species Reactivity
Human, Mouse, Rat
Validated Applications
WB, IHC-P, IF-Cell
Target Molecular Weight
Predicted band size: 52 kDa
Positive Control
HeLa treated with 10ng/mL BMP4 for 1 hour cell lysate, NIH/3T3 treated with 10ng/mL BMP4 for 1 hour cell lysate, human testis tissue, C6 treated with 10ng/mL BMP4 for 1 hour cell lysate, HeLa cells treated with 10ng/mL BMP4 for 1 hour, NIH/3T3 cells treated with 10ng/mL BMP4 for 1 hour.
Conjugation
unconjugated
Clone Number
JE59-46
Product Features
Form
Liquid
Concentration
1 mg/mL.(The concentration of this product may be batch-dependent)
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage Buffer
PBS (pH7.4).
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
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WB
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1:1,000
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IHC-P
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1:1,000
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IF-Cell
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1:100
Target
Function
Smads (or SMADs) comprise a family of structurally similar proteins that are the main signal transducers for receptors of the transforming growth factor beta (TGF-B) superfamily, which are critically important for regulating cell development and growth. There are three distinct sub-types of Smads: receptor-regulated Smads (R-Smads), common partner Smads (Co-Smads), and inhibitory Smads (I-Smads). The eight members of the Smad family are divided among these three groups. Trimers of two receptor-regulated SMADs and one co-SMAD act as transcription factors that regulate the expression of certain genes. The R-Smads consist of Smad1, Smad2, Smad3, Smad5 and Smad8/9, and are involved in direct signaling from the TGF-B receptor. R/Co-Smads are primarily located in the cytoplasm, but accumulate in the nucleus following TGF-β signaling, where they can bind to DNA and regulate transcription. However, I-Smads are predominantly found in the nucleus, where they can act as direct transcriptional regulators.
Background References
1. Nakatsu D et al. BMP4-SMAD1/5/9-RUNX2 pathway activation inhibits neurogenesis and oligodendrogenesis in Alzheimer\'s patients\' iPSCs in senescence-related conditions. Stem Cell Reports. 2023 Mar
2. Nakajima T et al. Changes in Smad1/5/9 expression and phosphorylation in astrocytes of the rat hippocampus after transient global cerebral ischemia. J Chem Neuroanat. 2021 Apr
Subcellular Location
Cytoplasm, Nucleus, Mitochondrion.
UNIPROT
Synonyms
BSP-1 antibody
BSP1 antibody
HsMAD1 antibody
JV4-1 antibody
JV41 antibody
MAD homolog 1 antibody
MAD mothers against decapentaplegic homolog 1 antibody
Mad related protein 1 antibody
Mad-related protein 1 antibody
MADH1 antibody
ExpandImages
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☑ Cell treatment (CT)
Western blot analysis of Phospho-Smad1/5/9 (S463/S465/S467) on different lysates with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/1,000 dilution.
Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 10ng/mL BMP4 for 1 hour cell lysate
Lane 3: HeLa treated with 10ng/mL BMP4 for 1 hour cell lysate, then the membrane treated with λpp for 1 hour
Lane 4: NIH/3T3 cell lysate
Lane 5: NIH/3T3 treated with 10ng/mL BMP4 for 1 hour cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 52 kDa
Observed band size: 52 kDa
Exposure time: Lane 1-3: 3 minutes; Lane 4-5: 42 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA752027) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA752027) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
☑ Cell treatment (CT)
Western blot analysis of Phospho-Smad1/5/9 (S463/S465/S467) on different lysates with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/1,000 dilution.
Lane 1: C6 cell lysate
Lane 2: C6 treated with 10ng/mL BMP4 for 1 hour cell lysate
Lane 3: C6 treated with 10ng/mL BMP4 for 1 hour cell lysate, then the membrane treated with λpp for 1 hour
Lysates/proteins at 10 µg/Lane.
Predicted band size: 52 kDa
Observed band size: 52 kDa
Exposure time: 1 minute 18 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA752027) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
☑ Cell treatment (CT)
Immunocytochemistry analysis of HeLa cells treated with 10ng/mL BMP4 for 1 hour labeling Phospho-Smad1/5/9 (S463/S465/S467) with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
☑ Cell treatment (CT)
Immunocytochemistry analysis of NIH/3T3 cells treated with 10ng/mL BMP4 for 1 hour labeling Phospho-Smad1/5/9 (S463/S465/S467) with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA752027) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
Products with the same target and pathway
Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46]
Application: WB,IHC-P,IF-Cell
Reactivity: Human,Mouse,Rat
Conjugate: unconjugated
