WB
Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody 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.IHC
mmunohistochemical analysis of paraffin-embedded human large intestine tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 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.ICC/IF
Immunocytochemistry analysis of A549 cells labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody 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 at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) 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 (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.IF-P
Immunofluorescence analysis of paraffin-embedded mouse kidney tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).WB
Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody 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. Exposure time: 2 minutes; 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 at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.| Product Name | Phospho-Smad3 (S423 + S425) Recombinant Rabbit Monoclonal Antibody |
|---|---|
| Antibody Type | Primary Antibodies |
| Immunogen | Synthetic phospho-peptide corresponding to residues surrounding Ser423 and 425 of human Smad3. |
| Modification | p-S423 + S425 |
| Modification Notes | 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. |
| Clonality | monoclonal |
|---|---|
| Isotype | IgG |
| Host Species | Rabbit |
| Tested Applications | ICC/IFIF-PIHCWB |
| WB:1:2000-1:5000 IHC:1:200-1:500 ICC/IF:1:500 IF-P:1:200 |
|
| Species Reactivity | HumanMouse |
| Concentration | 1mg/ml |
| Purification | Affinity purified |
| Gene Symbol | Smad3 |
|---|---|
| Gene Synonyms | LDS3 mad3 LDS1C MADH3 JV15-2 hMAD-3 hSMAD3 HSPC193 HsT17436 |
| Gene Full Name | SMAD3 SMAD family member 3 [ Homo sapiens (human) ] |
| Gene Summary | The SMAD family of proteins are a group of intracellular signal transducer proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. The SMAD3 protein functions in the transforming growth factor-beta signaling pathway, and transmits signals from the cell surface to the nucleus, regulating gene activity and cell proliferation. This protein forms a complex with other SMAD proteins and binds DNA, functioning both as a transcription factor and tumor suppressor. Mutations in this gene are associated with aneurysms-osteoarthritis syndrome and Loeys-Dietz Syndrome 3. [provided by RefSeq, May 2022] |
| Target | 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. |
| Molecular Weight(MW) | 48kDa(Observed band size: 55kDa) |
| Sequence Similarities | Belongs to the dwarfin/SMAD family. |
| Cellular Localization | Cytoplasm, Nucleus. |
WB
Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody 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.
IHC
mmunohistochemical analysis of paraffin-embedded human large intestine tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 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.
ICC/IF
Immunocytochemistry analysis of A549 cells labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody 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 at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) 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 (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.
IF-P
Immunofluorescence analysis of paraffin-embedded mouse kidney tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).
WB
Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody 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. Exposure time: 2 minutes; 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 at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.| 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, A431, human large intestine tissue, mouse skin tissue, mouse kidney tissue. |
|---|---|
| Application Notes | WB:1:2000-1:5000 IHC:1:200-1:500 ICC/IF:1:500 IF-P:1:200 |
| Form | Liquid |
|---|---|
| Storage Instructions | Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles. |
| Storage Buffer | 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide. |
