MDL-1 activation promotes NFATc1 nuclear translocation and enhances osteoclast formation. (A) Gene expression of pooled (n = 3) hind paws treated as in Fig. 3D were harvested at day 4 and prepared for Q-PCR analysis. Data are representative of four experiments. (B) Bone marrow cells were cultured in plastic dishes in the presence of 10 ng/ml RANKL and MCSF. Anti–MDL-1, but not isotype control (or medium control; not depicted) increased the number multinuclear cells (left) and osteoclast size (right). Data are representative of at least four separate experiments. Bars, 200 µm. (C) MDL-1 regulates NFATc1 nuclear expression. C57BL/6 bone marrow cells were cultured with indicated growth factors and antibodies. Cells were cultured for 6 d and nuclear lysates prepared for Active Motif Nuclear NFATc1 colorimetric assay. Nuclear NFATc1 was detected by a plate-bound NFAT oligonucleotide consensus sequence (5′-AGGAAA-3′), a primary NFATc1-specific antibody, and a secondary HRP-conjugated mAb. Results are representative of two studies. * indicates significance (P < 0.01) as determine by ANOVA analysis.