Signaling pathway analysis following MCSF and RANKL treatment.

A. Osteoclast precursors at day 3 and mature osteoclasts at day 4 were serum-starved and treated with MCSF, RANKL or MCSF+RANKL as indicated for five minutes. Equal amounts of total protein were analyzed by western blotting for the indicated phospho (p) or total (t) proteins. These data are representative of two separate experiments. For each experiment, marrow cells from three mice were pooled and analyzed in three replicate wells. The data are presented as the mean +/− SD from all replicate wells. B. Confocal images of the effect of RANKL and MCSF treatment on phospho- NFATc1 and total NFATc1, respectively. These data are representative of two separate experiments. C. Quantitative nuclear NFATc1 was calculated at T0 and T5′ after RANKL and MCSF treatment. Precursors were cultured with MCSF and RANKL as in Figure 1. On day 3, the cells were rinsed and serum starved for 1 hour prior to ether fixing (T0) or 5 minutes (T5′) of treatment with M-CSF and RANKL. These data are representative of two separate experiments and each experiment is analyzed in three replicate wells. D. Loss of TIEG1 expression results in decreased DC-STAMP expression in pre-fusion day 3 precursors compared to WT day 3 precursors. These data are representative of two separate experiments. For each experiment, marrow cells from three mice were pooled and analyzed in three replicate wells. The data are presented as the mean +/− SD from all replicate wells.