Horseradish peroxidase conjugated anti rabbit 1 1000

Protein extracts were prepared from E7 BPs treated with Bmp7 for 15 or 30 minutes at room temperature. Tissue was treated with RIPA buffer (SIGMA Aldrich) containing protease (Complete mini; Roche) and phosphatase (SIGMA Aldrich) inhibitor tabletsat 4 °C. Tissue was left on ice for 2 hrs with periodic vortexing. Tissue lysates were subsequently denatured at 85 °C for 5 minutes with NuPAGE LDS sample loading buffer (Invitrogen) and β-mercaptoethanol (SIGMA Aldrich). Proteins were then separated using SDS-PAGE on a 4-12% gradient Bis-Tris gel (Invitrogen) and subsequently transferred to nitrocellulose membranes (Invitrogen) over night at 4 °C. The primary antibodies and dilutions used were as follows: Rabbit anti-Tak1 (1:1000, Cell Signaling), Rabbit anti-phospho-Tak1 (1:1000, Cell Signaling), rabbit anti-Smad 1/5/8 (1:1000, Cell Signaling), rabbit anti-phospho Smad 1/5/8 (1:1000, Cell Signaling), mouse anti-β-Actin (1:5000, Sigma Aldrich). Horseradish peroxidase-conjugated anti-rabbit (1:1000) or anti-mouse (1:5000) IgGs were used as secondary antibodies (GE Healthcare). Specific bands were visualized by chemiluminescence using the SuperSignal^® West Pico Chemiluminescenct Substrate kit (Thermo Scientific).