STAT3 signaling pathway was involved in the IFN-β–mediated inhibition of osteoclastogenesis. (A) BMMs were infected with viruses harboring empty vector (pSuper) or Jak1 siRNA construct (Jak1siRNA). At 1 day after viral infection, cells were stimulated with IFN-β (100 U/mL) in combination with RANKL (500 ng/mL) for the indicated times. Total-cell lysates were subjected to Western blotting using phosphospecific antibodies. The expression of each protein was examined by directly stripping and reprobing the same membrane used to detect the phosphoform of the protein with respective antibody. (B) Vector or Jak1siRNA-infected BMMs were stimulated with 100 U/mL IFN-β for 30 minutes. Nuclear extracts were prepared and assayed for the DNA binding activities of STAT3 as described in “Methods.” Excess unlabeled oligo DNA was added to confirm the specificity of assay. (C) BMMs were first infected with viruses harboring empty vector (pSuper) or STAT3 siRNA construct (pSuper-STAT3siRNA). (D) After 48 hours, total-cell lysates were examined for STAT3 expression by Western blotting. Antiactin blot was shown to ensure same loading between 2 lanes. (E) Cells in panel D were incubated with 10 U/mL IFN-β for 24 hours and further incubated with 100 ng/mL RANKL for 3 days in the presence of 30 ng/mL M-CSF. At the end of culture, cells were stained for TRAP activity. The number of TRAP⁺ multinuclear OCs was counted from 3 random fields under microscope. Data are means plus or minus SD and are representative of 3 independent experiments. Asterisks represent statistical difference (**P < .01). MR represents M-CSF plus RANKL. (F) WT or STAT1-deficient BMMs were stimulated with 100 U/mL IFN-β for 30 minutes. Nuclear extracts were prepared and assayed for the DNA binding activities of STAT3 as described in “STAT3 DNA binding assay.” Excess unlabeled oligo DNA was added to confirm the specificity of assay. Data are means (± SEM) of 5 independent experiments. Asterisks represent statistical difference (*P < .05; **P < .01).