Recombinant Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903)

Blocking and diluting buffer: 5% NFDM/TBST.

Purified ab52903 staining Smad3 in Human stomach tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraffin and antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, Ph9.0). Samples were incubated with primary antibody at a 1/200 dilution. A ready to use rabbit specific IHC polymer detection kit HRP/DAP (ab209101). Hematoxylin was used as a counterstain. Nuclear and weakly cytoplasmic staining on human stomach without alkaline phosphatase treatment (image A). No signal can be detected when tissues were treated with alkaline phosphatase (image B).

Immunocytochemistry/Immunofluorescence analysis of A549 +/- TGFβ (5ng/ml, 24h) and A549 + TGFβ (5ng/ml, 24h) + Lamda phosphatase (LP) cells. Smad3 (phospho S423 + S425) was labelled with purified ab52903 at a dilution of 1/100 dilution, while Smad3 was labelled with ab207447 at a dilution of 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton X-100. ab150077 (goat anti-rabbit IgG Alexa Fluor^® 488) (1/1000) was used as the secondary antibody. The cells were co-stained with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) 1/200. Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

Blocking and dilution buffer: 5% NFDM/TBST.

Purified ab52903 staining Smad3 in Mouse kidney tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraffin and antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1/200 dilution. A ready to use rabbit specific IHC polymer detection kit HRP/DAP (ab209101). Hematoxylin was used as a counterstain. Nuclear and weakly cytoplasmic staining on mouse kidney without alkaline phosphatase treatment (image A). No signal can be detected when tissues were treated with alkaline phosphatase (image B).

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

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Representative IHC photomicrographs from an Environmental enteropathy (EE) duodenal biopsy showing p-SMAD3 staining (ab52903) in only the epithelium (arrows).

TGF-β1 signaling is impaired in NDRG1-silenced MEFs. PML^+/+ mouse embryonic fibroblasts (MEFs) were transfected with either CTL-siRNAs (A & B) or NDRG1-siRNAs (C & D) and induced with100 ng/ml TGF-β1. Immunofluorescent staining revealed intense nuclear staining for phosphorylated SMAD3 (SMAD3-P, ab52903) in CTL-siRNA treated MEFs (B) while only weak nuclear staining for MEFs treated with NDRG1-siRNA (D).

ab52903 staining Smad3 in mouse primary embryonic epicardial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and blocked with PBS + 1% BSA + 10% goat serum + 0.1% Triton X-100 for 1 hour at 20°C. Samples were incubated with primary antibody (1/100 in PBS + 1% BSA + 10% goat serum + 0.1% Triton X-100) for 16 hours at 4°C. An Alexa Fluor®488-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

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ab52903 staining Smad3 (phospho S423 + S425) in human TII Pneumocyte A549 cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton x100 before blocking with 3% BSA for 1 hour at RT. Samples were incubated with primary antibody (1/200: in 3% BSA in 1x PBST) for 24 hours at 4°C. A TRITC-conjugated goat polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution.

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Dot blot analysis of Smad 3 (phospho S423 + S425) phospho peptide (Lane 1), Smad 3 (phospho S423) phospho peptide (Lane 2), Smad 3 (phospho S425) phospho peptide (Lane 3) and Smad 3 non-phospho peptide (Lane 4) labelling Smad 3 (phospho S423 + S425) with ab52903 at a dilution of 1/1000. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000. Blocking and dilution buffer: 5% NFDM /TBST.