Table 1. Effect of inhibitors of protein kinases on nuclear accumulation of NFAT.

	Change in NFAT nuclear accumulation, %
Inhibited kinase	Control	Acidosis	RANKL	n
CK2	+22 ± 6*	-2 ± 4	0 ± 3	6
p38	+9 ± 6	0 ± 4	+19 ± 4*	6
GSK3β	+15 ± 12	+2 ± 2	+18 ± 5*	5
CK2 plus GSK3β	+38 ± 10*	+13 ± 6	+28 ± 4*	3
p38 plus GSK3β	+5 ± 23	+5 ± 4	+43 ± 11*†	4

CK2 was inhibited by using emodin (60 μM) or TBB (60 μM) (22); p38 kinase was inhibited by using SB202190 (10 μM) or PD169316 (10 μM); and GSK3β was inhibited by using LiCl (10 mM) (23). GSK3β and CK2 were inhibited with LiCl and either emodin or TBB. GSK3β and p38 were inhibited with LiCl and either SB202190 or PD169316. Osteoclasts in medium containing one or two kinase inhibitors or vehicle were incubated for 15 min at pH 7.6 (Control), at pH 7.0 (Acidosis), or with RANKL (1 μg/ml) at pH 7.6 (RANKL). Samples were then fixed, and NFAT localization was assessed by using immunofluorescence. For each condition, the difference in NFAT nuclear accumulation between inhibitor- and vehicle-treated osteoclasts was expressed as a percentage of vehicle-treated samples. Data are means ± SEM; n values indicate the number of independent experiments. Differences were assessed by Student's t test.

^*Significant difference compared with vehicle-treated samples (P < 0.001)

^†Significant difference compared with the effect of single inhibitor (P < 0.001)