Smad3 Recombinant Rabbit Monoclonal Antibody [SY25-01]

Overview

Product Name

Smad3 Recombinant Rabbit Monoclonal Antibody [SY25-01] - BSA and Azide free

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within Human Smad3 aa 181-230 / 425.

Species Reactivity

Human, Mouse, Rat

Validated Applications

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

Target Molecular Weight

Predicted band size: 48 kDa

Positive Control

HeLa cell lysate, A549 cell lysate, HL-60 cell lysate, NIH/3T3 cell lysate, bEnd.3 cell lysate, C6 cell lysate, Wild-type HaCaT whole cell lysate, A549, human lung carcinoma tissue, human breast carcinoma tissue, mouse liver tissue, mouse brain tissue, rat testis tissue, HeLa.

Conjugation

unconjugated

Clone Number

SY25-01

Reactivity Data

Tested

Verified (internally validated)

Published

Reported in literature (not internally validated)

Predicted

Predicted reactive (based on sequence homology)

Not recommended

Not recommended (failed internal validation)

	WB	IHC-P	FC	IF-Cell	IF-Tissue
human
mouse
rat

Product Features

Form

Liquid

Concentration

1mg/ml

Storage Instructions

Store at 2-8℃. Avoid freeze.

Storage Buffer

1*PBS (pH7.4).

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

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.

Background References

1. Dai X et al. SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model. Sci Rep 5:10180 (2015).

2. Chen JL et al. Development of novel activin-targeted therapeutics. Mol Ther 23:434-44 (2015).

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

SwissProt: P84025 Rat

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

This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Western blot analysis of Smad3 on different lysates with Rabbit anti-Smad3 antibody (ET1607-41) at 1/20,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: A549 cell lysate
Lane 3: HL-60 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: bEnd.3 cell lysate
Lane 6: C6 cell lysate

Lysates/proteins at 15 µg/Lane.

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

Exposure time: 1 minute 52 seconds;

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 (ET1607-41) at 1/20,000 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.
☑ Knockout (KO)

This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
All lanes: Western blot analysis of Smad3 with anti-Smad3 antibody (ET1607-41) at 1:1,000 dilution.
Lane 1: Wild-type HaCaT whole cell lysate (15 µg).
Lane 2: Smad3 knockout HaCaT whole cell lysate (15 µg).

ET1607-41 was shown to specifically react with Smad3 in wild-type HaCaT cells. NO band was observed when Smad3 knockout sample was tested. Wild-type and Smad3 knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1607-41, 1:1,000) was used in 5% BSA at room temperature for 2 hours. Goat anti-Rabbit IgG-HRP antibody at 1:10,000 dilution was used for 1 hour at room temperature.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of A549 cells labeling Smad3 with Rabbit anti-Smad3 antibody (ET1607-41) 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-Smad3 antibody (ET1607-41) 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue using anti-Smad3 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-41, 1/50) for 30 minutes 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-Smad3 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-41, 1/50) for 30 minutes 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded mouse liver tissue using anti-Smad3 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-41, 1/50) for 30 minutes 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Smad3 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-41, 1/50) for 30 minutes 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded rat testis tissue using anti-Smad3 antibody. 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 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-41, 1/200) for 30 minutes 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Smad3 antibody (ET1607-41) at 1/1,000 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 (ET1607-41) 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.
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Flow cytometric analysis of HeLa cells labeling Smad3.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1607-41, 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).
This data was developed using ET1607-41, the same antibody clone in a different buffer formulation.
Application: Immunofluorescence (IF-tissue)

Species: Human
Tissue: Kidney
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× TBST
Blocking: 10% normal goat serum + 1% Triton X-100 + 0.3 M Glycine in TBST, 30 minutes at room temperature.
Primary antibody: ET1607-41, 1/200, overnight at 4℃.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 1.5 hours at room temperature.