Figure 5.

Adseverin regulates osteoclastogenesis via the nuclear factor-κB (NF-κB) signaling pathway but not the mitogen-activated protein kinase (MAPK) signaling pathway. (a–c) After small interfering RNA (siRNA) transfection, bone marrow-derived macrophages (BMMs) were cultured with 30 ng ml⁻¹ macrophage-colony-stimulating factor (M-CSF) and 120 ng ml⁻¹ receptor activator of nuclear factor-κB ligand (RANKL) for 2 days. Cells were serum-starved for 4 h in α-modified Eagle's medium (α-MEM) and then stimulated with 600 ng ml⁻¹ RANKL. Stimulated cells were harvested after 0, 5, 15 and 30 min, and protein lysates were then prepared. Western blot analysis was performed using antibodies against the phosphoforms or total forms of the indicated proteins. (d) Cells were stimulated with RANKL as in a–c for 0, 30 and 60 min. Nuclear and cytoplasmic fraction proteins were prepared and subjected to western blot analysis.