Figure 5: NFAT activation in osteoblasts positively controls VCAM-1 expression. (a) Tibiae were harvested from 12-week-old mice and examined by immunohistochemistry with anti-VCAM-1 (brown) and counterstained with hematoxylin (blue). Negative control staining was performed by using normal rabbit IgG instead of primary antibody (top panel inset). Magnification, 400x. (b-c) Primary osteoblasts were harvested from calvariae of 1-day-old control and dnNFATOB mice and differentiated for 14 days in the presence of -glycerophosphate and ascorbic acid-2-phosphate to induce osteoblast differentiation. GFP control and ca-NFATc1-expressing MC3T3 E1 cells were cultured for 4 days. (b) Proteins were extracted from control and dnNFATOB differentiated primary osteoblasts, GFP control, and ca-NFATc1 MC3T3 E1 cells and separated by SDS-PAGE. Immunoblots were developed using antibodies against VCAM-1 and -actin. Immunoblots are representative of three independent experiments. (c–e) RNA was extracted from control and dnNFATOB differentiated primary osteoblasts ((c), left panel) and GFP control and ca-NFATc1 MC3T3 E1 cells ((c), right panel), and real-time PCR was performed for (c) VCAM-1, (d) CXCL12, and (e) IL-7 and -actin expression. Values were obtained from three independent experiments and represent the mean ± SD of VCAM-1, CXCL12, or IL-7 levels relative to -actin; * compared to control.