Fig. 1.

Release of sRANKL in conditioned media. (A) ELISA assays were performed to evaluate the concentrations of sRANKL and of the RANKL-decoy receptor osteoprotegerin in conditioned media from osteoblasts cultured for the indicated times, then the free sRANKL concentration was calculated on the basis of a RANKL/OPG 1:1 m ratio. (B, C) ELISA assays as described in (A) to calculate the free sRANKL concentration in conditioned media from (B) mouse bone marrow (BM) stromal cells and (C) mouse BM mesenchymal stem cells, cultured for 5 days, in comparison with mouse calvarial osteoblasts. (D) Real-time RT-PCR on RNA extracted from primary osteoblast cultures at the indicated times of culture. (E) Western blot analysis of sRANKL released in the osteoblast conditioned media pooled at the indicated times of culture. Numbers below the bands represent the relative arbitrary densitometric units. Data are the mean ± s.d. of three independent experiments. Statistics: unpaired Student's t test.