All experiments used BM mononuclear cells from 12-month-old mice. (A) IL-6 expression in OCLs formed in mouse BM cultures. IL-6 expression was assayed by Western blotting using anti–IL-6 antibody as describe in Methods. GAPDH was used as the loading control. Results are representative of 3 different experiments using 3 biological replicates for 4 genotype mice. (B) IL-6 levels in culture media. Purified OCLs (5 × 10⁴ cells/mL) from BM cultures derived from the 4 genotypes were cultured with 50 ng/mL of RANKL in αMEM-10% FCS for 72 hours, and CM were used fro ELISA assays of IL-6 and IGF1. The data are shown as mean ± SEM (4 technical replicates from the 4 genotypes). The data were analyzed using a 1-way ANOVA with Tukey test. *P < 0.01 as compared with WT mice. This assay was performed on 2 biological replicates and were similarly. (C) IGF1 levels in conditioned media. IGF1 was measured by ELISA as described in Methods. The data are shown as mean ± SEM (3 biological replicates the from the 4 genotypes). The data were analyzed using 1-way ANOVA with Tukey test. *P < 0.01 as compared with WT mice. This assay was performed in 2 individual experiments. (D) EphB2 expression in OCLs formed in mouse BM cultures. EphB2 expression was assayed by Western blotting using an EphB2 antibody as described in Methods. The expression levels of EphB2 were quantitated by ImageJ software (NIH). The basal ratio for every protein/loading control from OCLs of WT mice was set at 1.0. Results shown are from 2 biological replicates from the 4 genotypes.