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Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46]

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Specification

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Catalog# HA722566

Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46]

Application

  • WB

  • IHC-P

  • IF-Cell

Reactivity

  • Human

  • Mouse

  • Rat

Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Phospho-Smad1/5/9 (S463/S465/S467) Recombinant Rabbit Monoclonal Antibody [JE59-46]

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

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

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000

  • IHC-P

  • 1:1,000

  • IF-Cell

  • 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

SwissProt: Q15797 Human

SwissProt: Q99717 Human

SwissProt: O15198 Human

SwissProt: P70340 Mouse

SwissProt: P97454 Mouse

SwissProt: Q9JIW5 Mouse

SwissProt: O54835 Rat

SwissProt: P97588 Rat

SwissProt: Q9R1V3 Rat

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

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: HeLa treated with 10ng/mL BMP4 for 1 hour cell lysate<br />Lane 3: HeLa treated with 10ng/mL BMP4 for 1 hour cell lysate, then the membrane treated with λpp for 1 hour<br />Lane 4: NIH/3T3 cell lysate<br />Lane 5: NIH/3T3 treated with 10ng/mL BMP4 for 1 hour cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 52 kDa<br />Observed band size: 52 kDa<br /><br />Exposure time: Lane 1-3: 3 minutes; Lane 4-5: 42 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ 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 (HA722566) 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 (HA722566) 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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/1,000 dilution.<br /><br />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 ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) 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.

    Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-Phospho-Smad1/5/9 (S463/S465/S467) antibody (HA722566) 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 (HA722566) 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/1,000 dilution.<br /><br />Lane 1: C6 cell lysate<br />Lane 2: C6 treated with 10ng/mL BMP4 for 1 hour cell lysate<br />Lane 3: C6 treated with 10ng/mL BMP4 for 1 hour cell lysate, then the membrane treated with λpp for 1 hour<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 52 kDa<br />Observed band size: 52 kDa<br /><br />Exposure time: 1 minute 18 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ 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 (HA722566) 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 (HA722566) 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/100 dilution.<br /><br />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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) 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.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    ☑ 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 (HA722566) 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 (HA722566) 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/100 dilution.<br /><br />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 (<a href="/products/HA722566" style="font-weight: bold;text-decoration: underline;">HA722566</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) 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.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) 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 (HA722566) 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 (HA722566) 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"

Citation

Products with the same target and pathway

metafield image

Smad1/5/9 Rabbit Polyclonal Antibody

Application: WB,IHC-P,IF-Cell

Reactivity: Human,Mouse,Rat

Conjugate: unconjugated