Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HaCaT cells labelling Smad2 (phospho T8) + Smad3 (phospho T8) with ab254407 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and nuclear staining in HaCaT cells, while strong cytoplasmic and weak nuclear staining in HaCaT cells treated with calyculin A (100 nM) for 30 mins is observed. 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 cells labelling Smad2 (phospho T8) + Smad3 (phospho T8) with ab254407 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and nuclear staining in RAW 264.7 cells, while strong cytoplasmic and weak nuclear staining in RAW 264.7 cells treated with calyculin A (100 nM) for 30 mins is observed. 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.

• WB

Lab

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

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

The molecular weight observed is consistent with what has been described in the literature. (PMID : 25998442, 30666129).

Calyculin A is a known phosphatase inhibitor, which increased the level of pSmad2/3 (T8). The shifted band after treated with Calyculin A might be due to multiple phosphorylation events.

The down-regulation of pSmad2 (T8) is induced by TGF-beta1 treatment in HaCaT (PMID : 19201832).

Bands between 15-25kDa may be caused by degradation.

Exposure time : Lane 1, 2 : 26 secondsLane 3, 4 : 59 secondsLane 5, 6 : 3 minutes

All lanes:

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1:

HaCaT (human skin keratinocyte), whole cell lysate at 10 µg

Lane 2:

HaCaT - phosphatase treated membrane, whole cell lysate at 10 µg

Lanes 3 and 5:

HaCaT whole cell lysate at 20 µg

Lane 4:

HaCaT treated with 100nM calycin A for 30 min, whole cell lysate at 20 µg

Lane 6:

HaCaT treated with /ml TGF beta1 for 24h, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 55 kDa,60 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HaCaT (human skin keratinocyte) cells and 5µg of ab254407 [EPR23682-64]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

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

The molecular weight observed is consistent with what has been described in the literature. (PMID : 25998442, 30666129)

Exposure time : 59 seconds.

All lanes:

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg

Lane 2:

PC-12 - phosphatase treated membrane, whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

Smad2 (phospho T8) + Smad3 (phospho T8) was immunoprecipitated from 0.35 mg HaCaT (human skin keratinocyte), whole cell lysate with ab254407 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254407 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HaCaT (human skin keratinocyte), whole cell lysate 10 ug

Lane 2 : ab254407 IP in HaCaT whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab254407 in HaCaT whole cell lysate

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

Exposure time : 110 seconds

The molecular weight observed is consistent with what has been described in the literature. (PMID : 25998442, 30666129).

All lanes:

Immunoprecipitation - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407)

Observed band size: 55 kDa,60 kDa

false

• WB

Lab

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

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

The molecular weight observed is consistent with what has been described in the literature. (PMID : 25998442, 30666129).

Bands between 15-25kDa may be caused by degradation.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1:

Human liver cancer tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 55 kDa,60 kDa

false

• WB

Lab

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

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

Exposure time : 26 seconds.

All lanes:

Western blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 10 µg

Lane 2:

RAW264.7 - phosphatase treated membrane, whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (AB254407)

Dot blot analysis of Smad2 (phospho T8) + Smad3 (phospho T8) using ab254407 at 1/1000 (0.467 ug/ml) dilution followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Exposure time : 3 minutes.

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

Lane 1 : Smad2/3 peptide (aa 6-14)

Lane 2 : Smad2/3 (phospho T8) peptide (aa 2-10 )

Lane 3 : Smad2/3 peptide (aa 2-14)