Jak1 protein but not mRNA expression level was decreased during RANKL-induced osteoclast differentiation. (A) BMMs were cultured with 30 ng/mL M-CSF in the presence of indicated concentrations of RANKL for 24 hours. Lysates were subjected to Western blotting, and Jak1 expression was determined by enhanced chemiluminescence. Jak1 band densities were quantified as described in “Immunoprecipitation and immunoblotting” (right panel). (B) BMMs were cultured with 30 ng/mL M-CSF in the presence of 100 ng/mL RANKL for indicated times, and total-cell lysates were subjected to Western blotting using anti-Jak1 antibody (left panel). Jak1 expression was quantified as in panel A (right). (C) BMMs were cultured in the presence of 30 ng/mL M-CSF and 1 to 500 ng/mL RANKL for 3 days, and the formation of TRAP⁺ multinuclear OCs was observed. Cells were counted in 3 random fields under the microscope and the average number plus or minus SD was shown. Similar results were obtained from 2 independent experiments. (D) BMMs were cultured for 48 hours in the presence of 30 ng/mL M-CSF and 0 to 500 ng/mL RANKL, and total RNAs were isolated, reverse-transcribed, and subjected to quantitative real-time PCR analysis for Jak1. Jak1 expression levels were normalized by the levels of HPRT in each sample. (E) RNA samples from the cells cultured as in panel B were subjected to quantitative real-time PCR analysis. Western blot images are representative of at least 3 experiments with similar results. Values in graphs are expressed as means (± SD) from 3 (A,D) or 4 (B,E) experiments. *P < .05; **P < .01 versus control values.