FIGURE 7.

Effects of the expression of a 3BP2 cherubism mutant on Src activation and osteoclast differentiation in RAW264.7 cells. RAW264.7 cells were transfected with control vector, LZRS-V5–3BP2-IRES-GFP, or LZRS-V5–3BP2 R415P. A, GFP-positive cells were purified on a FACSAria cell sorter. B, sorted cells were stimulated for 5 days with sRANKL (40 ng/ml) and stained for TRAP activity. Morphology and multinucleated TRAP-positive cells were scored by microscopy as described above. Scale bars, 50 μm. The data are expressed as the means ± S.D. of three independent determinations. **, p < 0.01 versus shLacZ; ***, p < 0.001 versus shLacZ. C, purified cells were subjected to immunoblotting analysis using antibodies against V5 tag, 3BP2, phospho-Src Tyr⁴¹⁶. Protein loading was controlled with anti-Src and ERK2.