All lanes : 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
Lane 2 : HeLa grown in serum-free media overnight, then treated with 100 ng/ml Calyculin A (ab141784) for 15 minutes, followed by Calyculin A removal and treatment with 100 ng/ml BMP2 for 30 minutes, whole cell lysate
Lane 3 : NIH/3T3 (mouse embryo fibroblast cell line) grown in serum-free media overnight, whole cell lysate
Lane 4 : NIH/3T3 cultured in serum-free media overnight, then treated with 50 ng/ml BMP2 for 30 minutes, whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

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

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

Blocking/Dilution buffer: 5% NFDM/TBST.

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).

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.

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.