FIGURE 5.

Ectopic expression of active SrcY527F protein partially rescues RANKL-induced osteoclast formation in the absence of 3BP2 in RAW264.7 cells. A, 3BP2 interacts with c-Src, Syk, Vav1, and Cbl in resting RAW264.7 cells. The cells were lysed at 1 × 10⁸ cells/ml in ice-cold lysis buffer (1% Triton X-100 in 150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0.1% SDS, 0.1% sodium deoxycholate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) for 30 min on ice. Cleared lysates from 1 × 10⁷ cells were incubated with nonrelevant goat antibodies (NR) or sheep anti-3BP2 antibodies for 3 h at 4 °C followed by incubation with protein G-Sepharose beads for 1 h. After three washes, the immunoprecipitates were immunoblotted using antibodies against 3BP2, c-Src, Syk, Vav1, and Cbl. WCL, whole cell lysate. B, shLacZ and sh3BP2 cells were transfected with pLNCX SrcY527F retroviral construct or control vector, together with enhanced GFP expression vector. After 48 h, the GFP-positive cells were purified on a FACSAria cell sorter and subjected to immunoblotting analysis using antibodies against 3BP2, phospho-Src, phospho-Syk, and phospho-ERK1/2. Protein loading was controlled with anti-ERK2 immunoblot. Cells purified as indicated above were cultured for 4 days with sRANKL (40 ng/ml) and stained for TRAP activity. C and D, morphology (C) and multinucleated TRAP-positive cells (D) were assessed and scored by microscopy. Scale bars, 50 μm. TRAP+ multinucleated cell number and nuclei/osteoclast were counted. The data are expressed as the means ± S.D. of three equivalent wells and are representative of three independent experiments. ***, p < 0.001 versus shLacZ.