3.2. In-Cell Western for phosphorylated SMAD 1/5/8

Grow adherent cells to confluence in a tissue-culture treated clear bottom, white walled 96-well plate. Seed the suspension of cells, in 100 μL 10% FBS media per well, and allow cells to achieve confluence at 37°C and 5% CO₂ with humidification (see Note 1).

Wash confluent cells with 1X PBS in a sterile manner, and serum-starve cells overnight in 45 μL 1% FBS media per well.

Add 5 μL of varying dilutions of inhibitor compounds in PBS or vehicle control, and incubate for 10–30 min at 37°C and 5% CO₂ (see Note 7,8), being careful not to allow plate to cool.

Add BMP/TGF-β ligands in a volume of 5.5 μL to wells and incubate for 30min at 37°C and 5% CO₂, being careful not to allow plate to cool.

Remove supernatant and add 50 μL ice-cold methanol. Incubate at room temperature for 15 minutes.

Remove methanol and add 50 μL 0.5% glutaraldehyde in PBS. Incubate at room temperature for 15 minutes.

Wash in plate washer 4 times with 200 μL PBS or equivalent.

Block with 200 μL per well 2% BSA in PBS for 1 hour at room temperature on gentle rotator.

Remove blocking solution and add 50 μL per well primary antibody (rabbit mAb anti-p-SMAD 1/5/8), diluted 1:400 in 2% BSA in PBS. Incubate overnight at 4°C with gentle rotation or rocking.

Wash in plate washer 3 times with 200 μL PBS or equivalent

Add 50 μL per well secondary antibody (HRP-anti-rabbit IgG, Cell Signaling), diluted 1:400 in 2% BSA in PBS. Incubate 1 hour at room temperature on rotator

Wash plate in plate washer 4 times with 200 μL PBS or equivalent

Add 30 μL ECL reagent and read plate in a sensitive microplate luminometer immediately. Do not let the plate develop more than 2–3 minutes prior to reading.