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. 2015 Aug 14;290(39):23738–23750. doi: 10.1074/jbc.M115.667535

FIGURE 8.

FIGURE 8.

Inactivation of motif 2 and motif 3 drastically impairs the ability of RANK to up-regulate NFATc1 expression and abrogates the capacity of TNF/IL-1 to activate the expression of osteoclast genes. A, effects of inactivation of motif 2 and motif 3 on NFATc1 expression. BMMs expressing GFP, hFas-WT (WT) or hFas-M23 (M23) were treated with M-CSF (44 ng/ml) and α-Fas (100 ng/ml) for 3 days. The expression of NFATc1 was assessed by Western blot analysis using β-actin as a loading control. BMMs expressing GFP or WT were used as negative and positive controls, respectively. B, effects of inactivation of motif 2 and motif 3 on the expression of osteoclast genes. BMMs expressing GFP, hFas-WT, or hFas-M23 were treated with M-CSF (44 ng/ml) and α-Fas (10 ng/ml), M-CSF (44 ng/ml) and α-Fas (10 ng/ml) plus TNF (5 ng/ml), or M-CSF (44 ng/ml) and α-Fas (10 ng/ml) plus IL-1 (5 ng/ml) for 3 days. BMMs expressing GFP or WT were used as negative and positive controls, respectively. Gene expression was determined by semiquantitative RT-PCR using GAPDH as a loading control.