Figure 3: 5-HT₆R suppresses BMP-2 signaling by Jab1 to inhibit osteoblast differentiation.

(A,B) Primary calvarial osteoblasts from 1-day-old ICR mice were transfected with negative control siRNA or Jab1 siRNA for 24 h, further incubated in serum-free medium for 24 h, and then treated with 100 ng/mL rh-BMP2 for the times indicated. The levels of p-Smad1/5/8, Smad1/5/8, Jab1, and β-actin were analyzed by Western blot (A). The line graph data are expressed as relative fold-changes from each control (B). (C) Cells were cultured in OS with ST1936 for 3 days, and were immunoprecipitated with an anti-mouse antibody and mouse anti-Jab1 antibody. The immune complexes and cell lysates were analyzed by immunoblotting with rabbit anti-Jab1, anti-Runx2, and anti-Smad1/5/8 antibodies. (D) After Cells were transfected with negative control siRNA or Jab1 siRNA for 24 h, the cells were cultured in OS with ST1936 for 3 days. Protein levels of Runx2, Jab1, and β-actin were analyzed by Western blot. (E,F) Cells were transfected with control siRNA or Jab1 siRNA for 24 h, and then cultured in OS with ST1936 for 5 days. ALP was evaluated via ALP staining (E) and ALP activity (F). (G–I) After Cells were transfected with negative control siRNA or Jab1 siRNA for 24 h, the cells were cultured in OS with 10 μM ST1936 for 7 days. The mRNA levels of ALP (G), OCN (H) and BSP (I) were analyzed by real-time PCR, and normalized to that of β-actin. Data shown represent the means ± SEM. *p < 0.05, vs. control. ^#p < 0.05, vs. OS. &, p < 0.05, con-siRNA vs. Jab1 siRNA with 10 μM ST1936.