All lanes : Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate
Lane 3 : Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/20000 dilution

Predicted band size: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-4: Merged signal (red and green). Green - ab280888 observed at 60 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

ab280888 Anti-Smad2 (phospho S467) antibody [EPR23681-40] was shown to specifically react with Smad2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255430 (knockout cell lysate (ab263833) was used.  Wild-type and Smad2 knockout samples were subjected to SDS-PAGE. 

ab280888 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

Dot blot analysis of Smad2 (phospho S467) using ab280888 at 1:1000 (0.524 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: Smad2 peptide (aa 458-467)
Lane 2: Smad2 (phospho S465) peptide (aa 458-467)
Lane 3: Smad2 (phospho S467) peptide (aa 458-467)
Lane 4: Smad2 peptide (phospho aa S465/S467) peptide (aa 458-467)

Exposure time: 3 minutes

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

Immunohistochemical analysis of paraffin-embedded human stomach tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human stomach without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling Smad2 (phospho S467) with ab280888 at 1/50 (10.48 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).

Confocal image showing mainly nuclear staining in HeLa cells treated with TGF-β1 (20 ng/ml) for 15 mins.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes (Red)/ Untreated control (Green) cells labelling Smad2 (phospho S467) with ab280888 at 1/5000 dilution (0.01µg)/ red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in HeLa (PMID:24959295).

Smad2 (phospho S467) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate 10 µg with ab280888 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280888 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate 10 µg

Lane 2: ab280888 IP in HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280888 in HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate

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

Exposure time: 15 seconds.

All lanes : Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate at 10 µg
Lane 2 : HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate (phosphatase treated membrane) at 10 µg
Lane 3 : HeLa whole cell lysate at 20 µg
Lane 4 : HeLa treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 µg

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

Predicted band size: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature. (PMID: 24959295)

The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in HeLa (PMID:24959295).

Exposure time: 3 minutes

All lanes : Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 and 50 µM MG-132 for 15 minutes, whole cell lysate
Lane 2 : NIH/3T3 treated with 20ng/ml TGF beta1 and 50 µM MG-132 for 15 minutes whole cell lysate (phosphatase treated membrane)
Lane 3 : NIH/3T3 whole cell lysate
Lane 4 : NIH/3T3 treated with 20ng/ml TGF beta1 and 50 µM MG-132 for 15 minutes, whole cell lysate

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: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature. (PMID: 24347165)

The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in NIH/3T3 (PMID: 24347165)

Exposure time: 3 minutes

Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human colon carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse cardiac muscle without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat lung tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat lung without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling Smad2 (phospho S467) with ab280888 at 1/50 (10.48 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).

Confocal image showing mainly nuclear staining in NIH/3T3 cells treated with TGF-β1 (20 ng/ml) for 15 mins.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 for 15 minutes (Red)/ Untreated control (Green) cells labelling Smad2 (phospho S467) with ab280888 at 1/5000 dilution (0.01µg)/ red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in NIH/3T3 (PMID: 24347165).

Smad2 (phospho S467) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate 10 µg with ab280888 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280888 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 treated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate 10 µg

Lane 2: ab280888 IP in NIH/3T3 treated with 20ng/mltreated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280888 in NIH/3T3 treated with 20ng/mltreated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate

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

Exposure time: 32 seconds.