Western blotting analysis of BMP-2, Runx-2, OPN, and ALP

After 7 days in differentiation medium with osteogenic induction, two cell groups (miR-93-5p NC group and miR-93-5p mimic group) were seeded on 60 mm plastic dishes (WHB) and cultured for 7 days in osteogenic differentiation medium. Total protein was isolated using RIPA buffer. Proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane for 1.5 h at 4°C. Membranes were blocked with 5% milk in TBST for 2 h at room temperature and incubated with primary antibodies against BMP-2 (1:200, Santa Cruz, USA), OPN (1:200, Santa Cruz, USA), Runx-2 (1:200; Santa Cruz, USA), ALP (1:100, Boster, China), Osterix (1:200, Santa Cruz, USA), or GAPDH (1:3000, Tianjin Sungene, China) at 4°C overnight. Membranes were incubated with HRP-conjugated secondary antibody (1:2000, Boster, China) for 1 h at room temperature, followed by scanning with X-ray film. The integrated intensity for each detected band was then determined with ImageJ, v.1.46.