Azure 300 chemiluminescent imager

Bovine, porcine, and chicken MSCs and C2C12 cells were lysed using RIPA buffer containing 1% protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and total protein concentrations were quantified using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (50 μg) were subjected to 8% or 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were then blocked with BSA (Bovine serum albumin) or 3% skim milk in TBS (Tris-buffered saline) containing 0.1% Tween 20 for 1 h and incubated with target protein-specific primary antibodies [MYOD (1:400), MYOG (1:400), MYH (1:400), MTSN (1:400), SMAD2 (1:400), phosphorylated SMAD2 (1:400), ACVR2B (1:400), or ꞵ-actin (1:400)] in TBS containing 1% skim milk or BSA overnight at 4 °C. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse or anti-rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h and detected using the Dyne ECL Pico Plus Western Blotting Detection Kit (Dyne Bio, Seongnam, South Korea). Band images were analyzed using an Azure 300 chemiluminescent imager (Azure Biosystems, Dublin, CA, USA).