Fig. 5.

RANKL expression is increased in TRAF3 cKO osteoblastic cells. a Calvarial pre-OBs and spleen cells isolated from 7-day-old WT or cKO pups, co-cultured with 10⁻⁸ M 1,25(OH)₂Vitamin D₃ −/+ RANK:Fc (1 µg/ml) for 7 days and TRAP-stained. b Osteoclast numbers per well (Oc.N/well) in (a) were counted. Mean ± SD (n = 4 biologically independent samples; ^#p < 0.05, **p < 0.01). c WB of TRAF3, OPG, RANKL, and GAPDH in protein lysates from tibial metaphyseal bone from 3- and 9-m-old WT and cKO mice. d Densitometry analysis of RANKL/OPG protein ratio in (c). Mean ± SD (n = 3 biologically independent samples; *p < 0.05). e RANKL mRNA expression in BdMPCs from 3- and 9-m-old WT and cKO mice. Mean ± SD (n = 3 biologically independent samples; *p < 0.05, **p < 0.01). f BdMPCs from 3-m-old WT and cKO mice treated with TGFβ1 (1 ng/ml) for 0, 0.5, and 8 h. WB of RelA, RelB, HDAC2, and GAPDH in nuclei and cytoplasm. g Scheme for mouse RANKL promoter analysis showing putative κB binding sites and primer design to test binding sites. h, i Sheared chromatin from WT and cKO BdMPCs was used to perform DNA IP using h RelA, i RelB Abs or IgG control. Real-time PCR performed using designed primers that contain the putative κB binding sites 1, 2, 3, or an unrelated site, normalized to the input. Mean ± SD; n = 3 biologically independent samples; *p < 0.05. j Sheared chromatin from WT (W) and cKO (K) BdMPCs used to perform DNA IP using RelA, RelB, or IgG control Abs. PCR performed using designed primers that contain the putative κB binding sites 1, 2, 3, and an un-related site. All analyses done using one-way ANOVA with Tukey’s post-hoc test. All the in vitro experiments repeated twice with similar results