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. 2023 Dec 5;53(1):10. doi: 10.3892/ijmm.2023.5334

Figure 3.

Figure 3

Effect of MT RANKL on the AKT signaling cascade. (A) TRAP staining in the presence of WT RANKL (75 ng/ml) or MT RANKL (75 ng/ml) in BMDMs pretreated with MK2206 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 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 MK2206 pretreatment. GAPDH was used as a loading control. (D) Densitometric value of p-GSK-3β/GSK-3β and p-AKT/AKT determined by western blot analysis. 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; MT, mutant; N.S., not significant; p-, phosphorylated; RANKL, receptor-activated nuclear factor-κB ligand; siRNA, small interfering RNA; TRAP, tartrate-resistant acid phosphatase; WT, wild-type.