FIGURE 6.

Complementation of osteoclast formation by ectopic expression of constitutively active NFATc1 in 3BP2 knockdown cells. shLacZ and sh3BP2 cells were transfected with control vector or pRV-HA-caNFAT2-IRES-GFP vector encoding HA-tagged constitutively active caNFATc1 (caNFAT). After 48 h, the GFP-positive cells were purified on a FACSAria cell sorter. A, purified cells were subjected to immunoblotting analysis using antibodies against HA tag. Protein loading was controlled with anti-ERK2 immunoblot. The cells purified as indicated above were then cultured for 4 days in the presence of sRANKL (40 ng/ml) and stained for TRAP activity. B and C, morphology (B) and multinucleated TRAP-positive cells (C) were scored by microscopy. The cells with more than three nuclei were counted multinucleated cells, and the nuclei/osteoclast were counted. The data are expressed as the means ± S.D. of three independent determinations. **, p < 0.01 versus shLacZ; ***, p < 0.001 versus shLacZ.