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Phospho-Smad3 (S423 + S425) Recombinant Rabbit Monoclonal Antibody [ST0493]

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Specification

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Catalog# ET1609-41

Phospho-Smad3 (S423 + S425) Recombinant Rabbit Monoclonal Antibody [ST0493]

Application

  • WB

  • IF-Cell

  • IF-Tissue

  • IHC-P

Reactivity

  • Human

  • Mouse

BSA and Azide free
Conjugation

  • unconjugated

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

Overview

Product Name

Phospho-Smad3 (S423 + S425) Recombinant Rabbit Monoclonal Antibody [ST0493]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser423 and 425 of human Smad3.

Product Specificity

Phospho-SMAD3 (S423/S425) (ST0493) Rabbit mAb detects endogenous levels of SMAD3 only when dually phosphorylated at serines 423 and 425, and may detect SMAD2 phosphorylated at serines 465 and 467.

Species Reactivity

Human, Mouse

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P

Molecular Weight

Predicted band size: 48 kDa

Positive Control

A549 treated with 5ng/mL TGF-beta1 for 24 hours whole cell lysate, C2C12 treated with 5ng/mL TGF-beta1 for 24 hours whole cell lysate, HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate, A549, human large intestine tissue, mouse skin tissue, mouse kidney tissue.

Conjugation

unconjugated

Clone Number

ST0493

RRID

AB_3069851

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

  • 1:2,000-1:5,000

  • IF-Cell

  • 1:500

  • IF-Tissue

  • 1:200

  • IHC-P

  • 1:200-1:1,000

Target

Function

Smad proteins, the mammalian homologs of the Drosophila Mothers against dpp (Mad) have been implicated as downstream effectors of TGFβ/BMP signaling. Smad1 (also designated Madr1 or JV4-1), Smad5 and mammalian Smad8 (also designated Smad9 or MADH6) are effectors of BMP2 and BMP4 function while Smad2 (also designated Madr2 or JV18-1) and Smad3 are involved in TGFβ and activin-mediated growth modulation. Smad4 (also designated DPC4) has been shown to mediate all of the above activities through interaction with various Smad family members. Smad6 and Smad7 regulate the response to activin/TGFβ signaling by interfering with TGFβ-mediated phosphorylation of other Smad family members. Human Smad3 is subject to phosphorylation by TGFβ on specific amino acid residues, including Ser 208.

Background References

1. Marino FE et al. The inhibin/activin signalling pathway in human gonadal and adrenal cancers. Mol Hum Reprod 20:1223-37 (2014).

2. Tobar N et al. c-Jun N terminal kinase modulates NOX-4 derived ROS production and myofibroblasts differentiation in human breast stromal cells. BMC Cancer 14:640 (2014).

Sequence Similarity

Belongs to the dwarfin/SMAD family.

Post-translational Modification

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.

UNIPROT

SwissProt: P84022 Human

SwissProt: Q8BUN5 Mouse

Synonyms

DKFZP586N0721 antibody

DKFZp686J10186 antibody

hMAD 3 antibody

hMAD-3 antibody

hSMAD3 antibody

HSPC193 antibody

HST17436 antibody

JV15 2 antibody

JV15-2 antibody

JV152 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution.<br /><br />Lane 1: A549 whole cell lysate<br />Lane 2: A549 treated with 5ng/mL TGF-beta1 for 24 hours whole cell lysate<br />Lane 3: C2C12 whole cell lysate<br />Lane 4: C2C12 treated with 5ng/mL TGF-beta1 for 24 hours whole cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br /><br />Predicted band size: 48 kDa<br />Observed band size: 55 kDa<br /><br />Exposure time: 2 minutes; ECL: K1802;<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/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were 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-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution.

    Lane 1: A549 whole cell lysate
    Lane 2: A549 treated with 5ng/mL TGF-beta1 for 24 hours whole cell lysate
    Lane 3: C2C12 whole cell lysate
    Lane 4: C2C12 treated with 5ng/mL TGF-beta1 for 24 hours whole cell lysate

    Lysates/proteins at 15 µg/Lane.

    Predicted band size: 48 kDa
    Observed band size: 55 kDa

    Exposure time: 2 minutes; ECL: K1802;

    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 (ET1609-41) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were 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.

  • Immunocytochemistry analysis of A549 cells labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/500 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-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/500 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.

    Immunocytochemistry analysis of A549 cells labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/500 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-Smad3 (S423 + S425) antibody (ET1609-41) at 1/500 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 />Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/2,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate<br />Lane 3: A549 cell lysate<br />Lane 4: A549 treated with 5ng/mL TGF-beta1 for 24 hours cell lysate<br />Lane 5: NIH/3T3 cell lysate<br />Lane 6: NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 48 kDa<br />Observed band size: 55 kDa<br /><br />Exposure time: 2 minutes; 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/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/2,000 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-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/2,000 dilution.

    Lane 1: HeLa cell lysate
    Lane 2: HeLa starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate
    Lane 3: A549 cell lysate
    Lane 4: A549 treated with 5ng/mL TGF-beta1 for 24 hours cell lysate
    Lane 5: NIH/3T3 cell lysate
    Lane 6: NIH/3T3 starved overnight then treated with 10ng/mL TGF-β1 for 30 minutes cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 48 kDa
    Observed band size: 55 kDa

    Exposure time: 2 minutes; 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 (ET1609-41) at 1/2,000 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 large intestine tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/200 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/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/200 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 large intestine tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/200 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 (ET1609-41) at 1/200 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 mouse skin tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/500 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/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/500 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 mouse skin tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/500 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 (ET1609-41) at 1/500 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 mouse kidney tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</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/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</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 mouse kidney tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) 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 (ET1609-41) 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.

  • Immunofluorescence analysis of paraffin-embedded human large intestine tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/200 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. 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. Nuclei were counterstained with DAPI (blue).

    Immunofluorescence analysis of paraffin-embedded human large intestine tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/200 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1609-41, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

  • Immunofluorescence analysis of paraffin-embedded mouse kidney tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>) at 1/200 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (<a href="/products/ET1609-41" style="font-weight: bold;text-decoration: underline;">ET1609-41</a>, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. 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. Nuclei were counterstained with DAPI (blue).

    Immunofluorescence analysis of paraffin-embedded mouse kidney tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/200 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1609-41, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

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