Phospho-Smad3 (S423 + S425) Recombinant Rabbit Monoclonal Antibody [ST0493]

☑ Cell treatment (CT)

Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) 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; ECL: K1802;

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 (ET1609-41) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were 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.

☑ Cell treatment (CT)

Western blot analysis of Phospho-Smad3(S423/S425) on K562 cell lysates.

Lane 1: K562 cells, whole cell lysate, 10ug/lane
Lane 2: K562 cells treated with 2.8ug/ul lambda-PP for 30 minutes, whole cell lysates, 10ug/lane

All lanes :
Anti-Phospho-Smad3(S423/S425) antibody (ET1609-41) at 1/2,000 dilution. Anti-GAPDH antibody (ET1601-4) at 1:10,000 dilution. Goat Anti-Rabbit IgG H&L (HRP) (HA1001) at 1/200,000 dilution.

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

Blocking and diluting buffer: 5% BSA.

Exposure time: 3 minutes 43 seconds

☑ Cell treatment (CT)

Western blot analysis of Phospho-Smad3 (S423 + S425) on different lysates with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) 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.

Predicted band size: 48 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 (ET1609-41) at 1/2,000 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.

Immunocytochemistry analysis of A549 cells labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) 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-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) 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.

Immunohistochemical analysis of paraffin-embedded human large intestine tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/200 dilution.

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 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-41) 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.

Immunohistochemical analysis of paraffin-embedded mouse skin tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/500 dilution.

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 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-41) at 1/500 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 kidney tissue with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/1,000 dilution.

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 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-41) 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.

Immunofluorescence analysis of paraffin-embedded human large intestine tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/200 dilution.

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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1609-41, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Immunofluorescence analysis of paraffin-embedded mouse kidney tissue labeling Phospho-Smad3 (S423 + S425) with Rabbit anti-Phospho-Smad3 (S423 + S425) antibody (ET1609-41) at 1/200 dilution.

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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1609-41, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).