All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/2000 dilution (purified)

Lane 1 : F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates
Lane 2 : F9 (Mouse embryonic testicular cancer epithelial cell) whole cell lysates. Then the membrane was incubated with phosphatase.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 48 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

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.

All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution (purified)

Lane 1 : A549 whole cell lysate
Lane 2 : A549 treated with 5ng/ml TGF-ß1 for 24 hours whole cell lysate
Lane 3 : A549 treated with 5ng/ml TGF-ß1 for 24 hours whole cell lysate, the membrane was incubated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/2000 dilution

Lane 1 : (A) HL-60 cell lysates at 10µg untreated
Lane 2 : (B) HL-60 cell lysates at 10µg treated with TGF.

Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?
Additional bands at: 45 kDa. We are unsure as to the identity of these extra bands.

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).

All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution

Lane 1 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad3 non-phospho peptide
Lane 2 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad3 (phospho S423/425) peptide
Lane 3 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad2 non-phospho peptide
Lane 4 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad2 (phospho S465/467) peptide
Lane 5 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad1 non-phospho peptide
Lane 6 : HL-60 (human acute promyelocytic leukemia) treated with TGF-ß whole cell lysates, plus Smad1 (phospho S463/465) peptide

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/2000 dilution

Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

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

All lanes : Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] (ab52903) at 1/1000 dilution

Lane 1 : Lysate prepared from untreated human A549 cells
Lane 2 : Lysate prepared from untreated human A549 cells for 30min
Lane 3 : Lysate prepared from TGF-ß1 cells at 10ng/ml for 30min
Lane 4 : Lysate prepared from TNF-a cells at 20ng/ml for 30min
Lane 5 : Lysate prepared from TGF-ß1 and TNF-a cells at above doses for 30min
Lane 6 : Blank DMEM media

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Donkey Anti-Rabbit IgG H&L (HRP) (ab16284)

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 1 hour

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

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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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).

Immunohistochemical analysis of Smad3 in paraffin embedded human liver carcinoma tissue using ab52903 at 1/100 dilution.

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 human 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.