Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (AB226821)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Smad1 (phospho S463 + S465) with ab226821 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Nuclear staining in hBMP2-treated NIH/3T3 cells. Cells were FBS-deprived overnight before treatment with 50 ng/ml hBMP2 for 30 minutes.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (AB226821)

Smad 1 (phospho S463 + S465) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryo fibroblast cell line) grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate with ab226821 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab226821 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : NIH/3T3 grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate 10 μg (Input).

Lane 2 : ab226821 IP in NIH/3T3 grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab226821 in NIH/3T3 grown in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (ab226821)

Predicted band size: 52 kDa

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• WB

Lab

Western blot - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (AB226821)

Exposure time :
Lanes 1 and 2 : 3 minutes.
Lanes 3 and 4 : 30 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (ab226821) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) grown in serum-free media overnight, whole cell lysate at 20 µg

Lane 2:

HeLa grown in serum-free media overnight, then treated with 100 ng/ml Calyculin A (<a href='/ko/products/biochemicals/calyculin-a-protein-phosphatase-inhibitor-ab141784'>ab141784</a>) for 15 minutes, followed by Calyculin A removal and treatment with 100 ng/ml BMP2 for 30 minutes, whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryo fibroblast cell line) grown in serum-free media overnight, whole cell lysate at 20 µg

Lane 4:

NIH/3T3 cultured in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 52 kDa

Observed band size: 60 kDa

true

• Dot

Supplier Data

Dot Blot - Anti-Smad1 (phospho S463 + S465) antibody [EPR20662-20] (AB226821)

Dot blot analysis of Smad1 (phospho S463 + S465) labeled with ab226821 at 1/1000 dilution.

Lane 1 : Smad1 (phospho S463/S465) peptide;

Lane 2 : Smad1 (phospho S463) peptide;

Lane 3 : Smad1 (phospho S465) peptide;

Lane 4 : Smad1 peptide (not phosphorylated);

Lane 5 : Smad5 (phospho S463/S465) peptide;

Lane 6 : Smad5 (phospho S463) peptide;

Lane 7 : Smad5 (phospho S465) peptide;

Lane 9 : Smad5 peptide (not phosphorylated).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

Based on sequence homology, this antibody cross-reacts with Smad5 (phospho S463/S465) and Smad9 (phospho S465/S467).