RBP-J prevents IRF-8 down-regulation by blocking TNF-induced Blimp1 expression. (A) Immunoblot analysis of the expression of Blimp1 and NFATc1 in Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs obtained at the indicated time points after stimulation with TNF. p38 was measured as a loading control. (B) Rbpj^ΔM/ΔM BMMs were transfected with Blimp1-specific siRNAs or nontargeting control siRNAs (Control), and treated without or with TNF for 24 h. Blimp1 and IRF-8 expression was assessed by immunoblot. β-Tubulin was measured as a loading control. (C) Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs were transfected with Blimp1-specific siRNAs or nontargeting control siRNAs (Control), and treated with TNF for 24 h. NFATc1 expression was assessed by immunoblot. p38 was measured as a loading control. (D and E) Osteoclastogenesis assays of Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs transfected with siRNA targeting Blimp1 mRNA or nontargeting control siRNAs (Control) in the presence of TNF for 4 d. TRAP staining is shown in D and quantitated in E. Bar, 50 µm. **, P < 0.01. (F) Model for the regulation of TNF-induced osteoclastogenesis by RBP-J. RBP-J functions as a central upstream regulator of the balance between activating and inhibitory pathways by suppressing c-Fos expression and preventing Blimp1-mediated down-regulation of IRF-8. Data are representative of at least three independent experiments (A–E).