Western blotting for SMAD 1/5/8

Whole ovaries were homogenised in a RIPA buffer (Sigma-Aldrich, Israel) containing phosphatase and protease inhibitors (Roche, Switzerland). Protein content was determined with a bicinchoninic acid reagent, and 25 μg of protein from each sample was loaded onto 12% SDS-polyacrylamide gels. After transferring proteins to nitrocellulose membranes, blots were probed with appropriate antibodies: Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser465/467) (D5B10) rabbit mAb (no. 13820), vinculin (E1E9V) rabbit mAb (no. 13901) from Cell Signaling Technology (USA); peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (111-035-003, Jackson ImmumoResearch Laboratories, USA) was used to detect proteins through enzymatic chemiluminescence substrate (Westar Nova 2.0, Cyanagen).