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. 2001 Jan 15;15(2):241–253. doi: 10.1101/gad.840301

Figure 6.

Figure 6

Figure 6

Figure 6

Figure 6

Figure 6

Src251 blocks osteoclast responses to RANKL but not to M-CSF. (A) Osteoclasts were differentiated in culture, then washed twice with PBS and left in either complete media or media plus RANKL overnight. Wells (in quadruplicate) were stained for TRAP activity either before (pre) or after treatment (media or RANKL), and TRAP-positive multinucleated cells were counted. (B) AKT activity in response to RANKL. Osteoclast cultures were serum starved, treated with RANKL for 10 min, lysed, and AKT-immunoprecipitated; and AKT kinase activity was evaluated using GST–GSK3α as a substrate. (C) WT, Src251, Src251-ΔSH3, and Src251-R175L osteoclasts were treated with RANKL overnight, as in A. Src251-ΔSH3 blocks cell survival in response to RANKL, paralleling its induction of osteopetrosis. (D) Osteoclasts from src−/− and Src251 mice both respond poorly to RANKL, but respond normally to M-CSF. Osteoclasts differentiated in culture were treated as above or cultured in media with M-CSF overnight. (E) Src251 does not block AKT activation in response to M-CSF. Osteoclast cultures were serum starved, then treated with M-CSF for 10 min, lysed, and immunoblotted with anti-pS473-AKT to monitor AKT activation.