Fig 4.
RANKL activates cytoskeleton-organizing molecules principally via c-Src MYR and SH2 domains. (A) c-Src−/− spleen-derived macrophages, transduced with either FLAG-tagged WT Src, ΔMYR, ΔSH3, or ΔSH2 were cultured with RANKL and M-CSF for 5 days on bone powder. Serum- and cytokine-starved cells were exposed to RANKL (100 ng/ml) for 20 min. Phosphorylated c-SrcY416 and FLAG in FLAG immunoprecipitates (IP) (A), phosphotyrosine (p-Y) and SLP-76 in SLP-76 immunoprecipitates (B), phosphotyrosine and Syk in Syk immunoprecipitates (C), and phosphotyrosine and Vav3 in Vav3 immunoprecipitates (D) were determined by immunoblotting. Rac1-GTP (E) and RhoA-GTP (F) were detected by pulldown assay, and total GTPase was detected by immunoblotting. Densitometric data are expressed relative to vector (Vect)/RANKL−. The experiments were repeated twice.