All experiments used 12-month-old mice. (A) Image of ALP staining of OB outgrowing cells from bone cultured for 14 days. Cells were stained ALP and staining was quantitated by ImageJ software (NIH). Results are representative of 3 biological replicates. (B) Expression of OB differentiation maker proteins. The expression of EphB4, osterix, and Col-1A were assessed by Western blotting using anti-EphB4 antibody, anti-osterix antibody, and anti–Col-1A antibody as described in Methods. β-Actin was used as the loading control. The expression levels of EphB4, osterix, and Col-1A were quantitated by ImageJ software (NIH). The basal ratio for every protein/loading control for OBs from WT mice was set at 1.0. Results are representative of 3 biological replicates. (C) OB differentiation in cocultures with MVNP-OCLs. OCLs (5 × 10⁴/well) derived from MVNP mice were scraped with a rubber policeman as described in Methods and were then replated and cultured with 50 ng/mL RANKL overnight. The OBs (1 × 10⁵/well) described above were plated on top of the OCLs the next day, and the cells were cocultured for 72 hours. The expression levels of EphB4, osterix, and Col-1A was determined by Western blotting using these antibodies as above. β-Actin was used as the loading control. The expression levels of EphB4, osterix, and Col-1A were quantitated by ImageJ software. The basal ratio of every protein/loading control for OBs from WT mice was set at 1.0. Results are expressed as the mean ± SEM (representative of 3 biological replicated for 4 genotype mice). The data were analyzed using 1-way ANOVA with Tukey test. *P < 0.01 as compared with each indicated group. These results of experiment were similarly in 2 different biological replicates.