Western Blot Analysis of BMP2 and Smad7

Cells were seeded in six-well plates and treated per the experimental design. Total protein was obtained after lysis, and the cleared lysates were denatured by boiling for 10 min with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer. The proteins were separated by electrophoresis with Tris-glycine gels and carefully transferred onto polyvinylidene difluoride (PVDF) membranes in the dark. Then, the PVDF membranes were blocked with 5% evaporated milk for 1 h and incubated overnight with primary antibodies against BMP2 (Abcam, USA) and Smad7 (Santa Cruz, USA). After washing, the membranes were probed with a fluorescently labeled secondary antibodiesy. The immune-reactive signals were detected using a Bio-Rad machine. In addition, the membranes were incubated with a monoclonal mouse anti-human β-actin (Abcam, USA) antibody as a loading control. The relative band intensity was measured using ImageJ analysis software [35 (link), 36 (link)].