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Phospho-Smad2 (S250) Recombinant Rabbit Monoclonal Antibody [SD207-1]

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Specification

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Catalog# ET1612-32

Phospho-Smad2 (S250) Recombinant Rabbit Monoclonal Antibody [SD207-1]

Application

  • WB

  • FC

  • IP

  • IHC-P

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

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

Overview

Product Name

Phospho-Smad2 (S250) Recombinant Rabbit Monoclonal Antibody [SD207-1]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser250 of Human Smad2 aa 221-270 / 467.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, FC, IP, IHC-P

Molecular Weight

Predicted band size: 52 kDa

Positive Control

HeLa cell lysate, HeLa treated with 200nM PMA for 30 minutes cell lysate, NIH/3T3 cell lysate, NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate, C6 cell lysate, C6 treated with 200nM PMA for 30 minutes cell lysate, HeLa, human lung cancer tissue, mouse testis tissue, rat testis tissue.

Conjugation

unconjugated

Clone Number

SD207-1

RRID

AB_3070105

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

  • 1:5,000

  • FC

  • 1:50-1:100

  • IP

  • 1-2μg/sample

  • IHC-P

  • 1:50

Target

Function

Smad proteins, the mammalian homologs of the Drosophila mothers against decapentaplegic (Mad), have been implicated as downstream effectors of TGFβ/BMP signaling. Smad1 (also designated Madr1 or JV4-1) and Smad5 are effectors of BMP-2 and BMP-4 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 proteins.

Background References

1. Ungefroren H et al. Rac1b negatively regulates TGF- 1-induced cell motility in pancreatic ductal epithelial cells by suppressing Smad signalling. Oncotarget 5:277-90 (2014).

2. Harazono Y et al. miR-655 Is an EMT-suppressive MicroRNA targeting ZEB1 and TGFBR2. PLoS One 8:e62757 (2013).

Sequence Similarity

Belongs to the dwarfin/SMAD family.

Tissue Specificity

Expressed at high levels in skeletal muscle, endothelial cells, heart and placenta.

Post-translational Modification

Phosphorylated on one or several of Thr-220, Ser-245, Ser-250, and Ser-255. In response to TGF-beta, phosphorylated on Ser-465/467 by TGF-beta and activin type 1 receptor kinases. TGF-beta-induced Ser-465/467 phosphorylation declines progressively in a KMT5A-dependent manner. Able to interact with SMURF2 when phosphorylated on Ser-465/467, recruiting other proteins, such as SNON, for degradation. In response to decorin, the naturally occurring inhibitor of TGF-beta signaling, phosphorylated on Ser-240 by CaMK2. Phosphorylated by MAPK3 upon EGF stimulation; which increases transcriptional activity and stability, and is blocked by calmodulin. Phosphorylated by PDPK1.; In response to TGF-beta, ubiquitinated by NEDD4L; which promotes its degradation. Monoubiquitinated, leading to prevent DNA-binding (By similarity). Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes. Ubiquitinated by RNF111, leading to its degradation: only SMAD2 proteins that are 'in use' are targeted by RNF111, RNF111 playing a key role in activating SMAD2 and regulating its turnover (By similarity).; Acetylated on Lys-19 by coactivators in response to TGF-beta signaling, which increases transcriptional activity. Isoform short: Acetylation increases DNA binding activity in vitro and enhances its association with target promoters in vivo. Acetylation in the nucleus by EP300 is enhanced by TGF-beta.

Subcellular Location

Cytoplasm, Nucleus.

UNIPROT

SwissProt: Q15796 Human

SwissProt: Q62432 Mouse

SwissProt: O70436 Rat

Synonyms

Drosophila, homolog of, MADR2 antibody

hMAD-2 antibody

HsMAD2 antibody

JV18 antibody

JV18-1 antibody

JV181 antibody

MAD antibody

MAD homolog 2 antibody

MAD Related Protein 2 antibody

Mad-related protein 2 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-Smad2 (S250) on different lysates with Rabbit anti-Phospho-Smad2 (S250) antibody (<a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/5,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: HeLa treated with 200nM PMA for 30 minutes cell lysate<br />Lane 3: NIH/3T3 cell lysate<br />Lane 4: NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 52 kDa<br />Observed band size: 55 kDa<br /><br />Exposure time: 30 seconds; ECL: K1801;<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/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/5,000 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-Smad2 (S250) on different lysates with Rabbit anti-Phospho-Smad2 (S250) antibody (ET1612-32) at 1/5,000 dilution.

    Lane 1: HeLa cell lysate
    Lane 2: HeLa treated with 200nM PMA for 30 minutes cell lysate
    Lane 3: NIH/3T3 cell lysate
    Lane 4: NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 52 kDa
    Observed band size: 55 kDa

    Exposure time: 30 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 (ET1612-32) at 1/5,000 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 />Western blot analysis of Phospho-Smad2 (S250) on different lysates with Rabbit anti-Phospho-Smad2 (S250) antibody (<a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/5,000 dilution.<br /><br />Lane 1: C6 cell lysate<br />Lane 2: C6 treated with 200nM PMA for 30 minutes cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 52 kDa<br />Observed band size: 55 kDa<br /><br />Exposure time: 2 minutes; ECL: K1801;<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/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/5,000 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-Smad2 (S250) on different lysates with Rabbit anti-Phospho-Smad2 (S250) antibody (ET1612-32) at 1/5,000 dilution.

    Lane 1: C6 cell lysate
    Lane 2: C6 treated with 200nM PMA for 30 minutes cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 52 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 (ET1612-32) at 1/5,000 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 />Phospho-Smad2 (S250) was immunoprecipitated from 0.2 mg HeLa treated with 100nM TPA for 30 minutes cell lysate with <a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a> at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using <a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a> at 1/5,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.<br /><br />Lane 1: HeLa treated with 100nM TPA for 30 minutes cell lysate (input)<br />Lane 2: <a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a> IP in HeLa treated with 100nM TPA for 30 minutes cell lysate<br />Lane 3: Rabbit IgG instead of <a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a> in HeLa treated with 100nM TPA for 30 minutes cell lysate<br /><br />Blocking/Dilution buffer: 5% NFDM/TBST<br />Exposure time: 35 seconds; ECL: <a href="/products/K1802" style="font-weight: bold;text-decoration: underline;">K1802</a>

    ☑ Cell treatment (CT)

    Phospho-Smad2 (S250) was immunoprecipitated from 0.2 mg HeLa treated with 100nM TPA for 30 minutes cell lysate with ET1612-32 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1612-32 at 1/5,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

    Lane 1: HeLa treated with 100nM TPA for 30 minutes cell lysate (input)
    Lane 2: ET1612-32 IP in HeLa treated with 100nM TPA for 30 minutes cell lysate
    Lane 3: Rabbit IgG instead of ET1612-32 in HeLa treated with 100nM TPA for 30 minutes cell lysate

    Blocking/Dilution buffer: 5% NFDM/TBST
    Exposure time: 35 seconds; ECL: K1802

  • Immunohistochemical analysis of paraffin-embedded human lung cancer tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (<a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/50 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/50 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 lung cancer tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (ET1612-32) at 1/50 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1612-32) at 1/50 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 testis tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (<a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/50 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/50 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 testis tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (ET1612-32) at 1/50 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1612-32) at 1/50 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 rat testis tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (<a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/50 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>) at 1/50 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 rat testis tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (ET1612-32) at 1/50 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1612-32) at 1/50 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.

  • Flow cytometric analysis of HeLa cells labeling Phospho-Smad2 (S250).<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET1612-32" style="font-weight: bold;text-decoration: underline;">ET1612-32</a>, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of HeLa cells labeling Phospho-Smad2 (S250).

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET1612-32, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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