C2C12 cells were seeded at 5K cells/well in a 96 well tissue culture microplate overnight. The next day cells were serum starved for 60 minutes, then treated with various concentrations of dorsomorphin for 30 mins. Subsequently, the cells stimulated with 50 ng/mL BMP-4 for 30 min. The medium was removed from the wells, and cells were lysed with 120 µl/well of Lysis Mix, with shaking for 10 min. The lysates were transferred to a PhosphoTracer assay plate and assayed for either total or phospho-SMAD1, using the standard protocol. Signal in the wells was determined using a plate reader.
Using the SMAD1 assay kits or Western blot, SMAD1 phosphorylation at Ser463/465 is detected in BMP4-treated C2C12 cells (+), compared with C2C12 cells treated with 50 µM dorsomorphin (-). Upon phosphorylation, SMAD1 levels decrease as SMAD1 becomes ubiquitinated.