TRAF6 levels and STAT1 signaling are intact in late pre-osteoclasts. (a) Untreated RAW cells were stimulated with IFN-γ for 5, 15 and 60 min, and then protein extracts were analyzed by immunoblotting as described in Materials and methods. Multiple signals were achieved by stripping and reprobing the same blot. TRAF6 expression increased with IFN-γ signaling. (b) RAW cells were cultured for 4 days in the presence of the indicated cytokines. Osteoclasts were observed in the receptor-activator of NFκB ligand (RANKL) group, while activated macrophages were observed in the RANKL + IFN-γ group similar to that described in the previous experiments. TRAF6 expression was remarkably consistent in these cells. (c) Nuclear extracts were prepared from untreated RAW cells and late pre-osteoclasts (treated with RANKL for 2 days) and immunoblotted with antiphospho-Stat1 antibodies to assess nuclear translocation in extracts obtained at the indicated time points following IFN-γ treatment. Late pre-osteoclasts translocated phospho (p)-Stat1 as well as untreated RAW cells.