RBP-J deficiency results in increased TNF-induced NFATc1 expression and function. (A) Quantitative real-time PCR analysis of mRNA expression of Nfatc1 in osteoclastogenic cell cultures from Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated with TNF for the indicated times. *, P < 0.05; **, P < 0.01. (B) Immunoblot analysis of NFATc1 expression in whole-cell lysates obtained from Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs at the indicated time points after stimulation with TNF. p38 was measured as a loading control. (C) Quantitative real-time PCR analysis of mRNA expression of Itgb3 (encoding integrin β3) in osteoclastogenic cell cultures from Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated with TNF for the indicated times. **, P < 0.01. (D) ChIP analysis of NFATc1 occupancy at the promoter of Nfatc1 in the Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated without or with TNF for 48 h. **, P < 0.01. (E) Quantitative real-time PCR analysis of expression of primary transcripts of Nfatc1 in osteoclastogenic cell cultures from Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated with TNF. **, P < 0.01. (F) ChIP analysis of polymerase II occupancy at the Nfatc1 locus in Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated without or with TNF for 24 h. Data are representative of at least three independent experiments (A–F). **, P < 0.01.