Fig. 2. JNK1 but not JNK2 is activated by RANKL in osteoclast progenitors. (A) The expression of JNK isoforms was analyzed in bone marrow monocytes (BMMs) isolated from wild-type, Jnk1^-/- and Jnk2^-/- mice and cultured for 3 days in the presence of M-CSF alone (20 ng/ml) (-) or M-CSF and RANKL (50 ng/ml) (+). The expression of JNK isoforms was determined by western blotting using an antibody that recognizes both JNK1 and JNK2. (B,C) Time-dependent activation of JNK by RANKL. M-CSF-dependent monocytes were isolated from wild-type, Jnk1^-/- and Jnk2^-/- mice and treated with RANKL (50 ng/ml) for the indicated times. The kinase activity was assayed following JNK immunoprecipitation using GST-N-terminal-c-Jun fusion protein as a substrate. The phosphorylation of GST-c-Jun was quantified after SDS PAGE separation and phosphoimager analysis. (D) JNK1 immunodepletion. Wild-type M-CSF-dependent monocytes were treated with RANKL (50 ng/ml) for 15 minutes. JNK1 and JNK2 were sequentially immunoprecipitated, and JNK activity was assayed in both fractions. (E) Activation of JNK2 in Jnk1^-/- BMMs. Wild-type and Jnk1^-/- M-CSF-dependent monocytes were exposed to UV irradiation (40 J/m²). Kinase activity was measured following JNK immunoprecipitation.

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