Effect of MT RANKL on the GSK-3β signaling cascade. (A) TRAP staining results in the presence of WT RANKL (75 ng/ml) or MT RANKL (75 ng/ml) in BMDMs pretreated with LiCl for 8 h before RANKL treatment. Magnification, ×100; scale bar, 20 μm. (B) Number of multinucleated TRAP-positive cells (≥3 nuclei). ***P<0.001. (C) Western blot analysis of the RANK and LGR4 signaling cascades. BMDMs were exposed to WT RANKL (2 μg/ml) or MT RANKL (2 μg/ml) for 0, 5, 10 and 15 min after 8 h with or without LiCl pretreatment. GAPDH was used as a loading control. (D) Densitometric value of p-GSK-3β/GSK-3β and p-AKT/AKT was determined by western blot analysis. The results are representative of three separate experiments that had comparable results. BMDM, bone marrow-derived macrophage; GSK-3β, glycogen synthase kinase-3β; LGR4, leucine-rich repeat-containing G-protein-coupled receptor 4; LiCl, lithium chloride; MT, mutant; N.S., not significant; p-, phosphorylated; RANKL, receptor-activated nuclear factor-κB ligand; TRAP, tartrate-resistant acid phosphatase; WT, wild-type.