Expression of osteoblast lineage markers and CSF-1R transcripts and effect of CSF-1 on tyrosine phosphorylation of CSF-1R in murine primary osteoblasts. A, Expression of osteoblast lineage markers. Total RNA was isolated from primary cultures in complete medium, and mRNA expression of the indicated genes was determined using RT-PCR. B, FACS analysis of CD45, CD11b, and F4/80 expression in osteoblasts (upper panel) and bone marrow control (lower panel). In histograms, a dotted line represents unstained cells, and solid and dashed lines represent antibody staining in trypsinized and untrypsinized cells, respectively. C, RT-PCR analysis of CSF-1 and CSF-1R mRNAs in osteoblasts (WT OB.) and RAW264.7 cells (control). Total RNA was isolated from cells, and RT-PCR was carried out using murine CSF-1, CSF-1R, or 18S (control) primer sets. D, CSF-1-induced tyrosine phosphorylation. Cells were placed in SF medium and incubated with or without rmCSF-1 (1–100 ng/ml) at the indicated time points before harvesting at 48 h. Western blot analysis of CSF-1R protein (upper panels, 100 μg/lane) and immunocomplex kinase assay (lower panels) are shown. E, CSF-1R mRNA (upper panel) and protein (lower panel) expression in osteoblast cell lines.