Figure 3.

Salubrinal downregulated P65 abundance and inhibited the RANKL-induced NF-κB signaling pathway. (A) Phospho-IκBα, IκBα, phospho-P65, and P65 expression levels were analyzed by Western blotting after stimulation with RANKL (30 ng/mL) for the indicated times in bone marrow-derived osteoclast precursors pretreated with Salubrinal (10 µM) for 3 h. P65 abundance in the nucleus and cytoplasm was analyzed by Western blotting (B) and immunofluorescence staining (C) after stimulation with RANKL (30 ng/mL) for 30 min in bone marrow-derived osteoclast precursors pretreated with Salubrinal (10 µM) for 3 h. (D) NF-κB signaling transcriptional activity was measured using dual-luciferase reporter assays. (E) P65 abundance in knee joints of CIA mice was detected by immunohistochemical staining. Data are shown as means ± SEM. ** p < 0.01.