Figure 1: Generation of dnNFATOB mice. (a) Representation of ColI-dnNFAT transgene construct. (b) DNA was extracted from tail biopsies and amplified by PCR for a 0.55 kb fragment of the ColI-dnNFAT transgene. Mice without ColI-dnNFAT were used as controls. The amplification of thyroid stimulating hormone-beta (TSH-) was used as a loading control. (c) Primary osteoblasts were harvested from calvariae of 1-day-old mice and differentiated 7–14 days. Brain tissue was removed from 12-week-old mice and homogenized. Nuclear proteins were used for immunoblotting with antibodies against NFATc1 and lamin C. Immunoblots are representative of three independent experiments (). (C = control; DN = dnNFATOB.) (d) Femora were harvested from 12-week-old control () and dnNFATOB () mice and examined by immunohistochemistry with anti-NFATc1 (brown), counterstained with hematoxylin (blue). Negative control staining was performed by using normal rabbit IgG instead of primary antibody (left panel inset). Magnification, 400x.