Constitutive activation of RBP-J signaling in myeloid osteoclast precursors suppresses NFATc1 expression and osteoclastogenesis. (A) Immunoblot analysis of NFATc1 in control and NICD1^M BMMs treated with RANKL for the indicated times. p38 was measured as a loading control. (B) Quantitation of TRAP-positive MNCs in cell cultures from control and NICD1^M BMMs treated with RANKL in the presence of M-CSF for 3 d. *, P < 0.05. (C) Quantitative real-time PCR analysis of Rbpj mRNA in control and NICD1^M BMMs transfected with siRNA targeting Rbpj or nontargeting control siRNAs (Control) for 24 h. **, P < 0.01. (D) Immunoblot analysis of NFATc1 in control and NICD1^M BMMs transfected with Rbpj or control siRNAs and stimulated with RANKL for 24 h. p38 was measured as a loading control. (E) Quantitation of TRAP-positive MNCs in cell cultures from control and NICD1^M BMMs transfected with Rbpj or control siRNAs and stimulated with RANKL for 3 d. *, P < 0.05; NS, no statistically significant difference. Data are representative of at least three independent experiments (A–E).