BMSCs seeded on coverslips were fixed in 4% paraformaldehyde and treated with 0.5% Triton X-100 for 15 min. After being rinsed twice with PBS, the cells were blocked with 5% BSA for 30 min at room temperature. Soon afterwards, the cells were incubated with primary antibodies [rabbit anti-Runx2 (1:100, Cell Signaling Technology), mouse anti-OPN (1:50, Santa Cruz Biotechnology), rabbit anti-phospho-Smad1/5/8 (1:100, Cell Signaling Technology), mouse anti-BMPRIB (1:50, Santa Cruz Biotechnology), rabbit anti-p38 MAPK (1:50, Cell Signaling Technology), mouse anti-TAB1 (1:50, Santa Cruz Biotechnology)] against the target proteins at 4 °C overnight. Secondary antibodies including CY3-conjugated goat anti-rabbit IgG (1:200, Boster, China) and FITC-labeled goat anti-mouse IgG (1:200, Boster, China) were used to bind the primary antibodies. The fluorescence images were acquired under a confocal microscope (Eclipse, NIKON, Japan).

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