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Fig. 4. Ncad
C expression in vivo retards osteoblast differentiation. (A) Calvaria cells (CLVC) were cultured in 24-well plates up to 3 weeks after confluence, and alkaline phosphatase activity was measured in the cell layer at weekly intervals, and normalized to cellular protein content. Enzyme activity was reduced in CLVC from newborn OG2-NcadC mice compared with wild-type (WT) cells at 1 and 2 weeks post-confluence. *P<0.001, t-test (mean±s.e.m.). (B) Von Kossa staining of CLVC cultures isolated from newborn OG2-NcadC transgenic (Tg) or wild-type (WT) mice, after 2 or 3 weeks in culture in the presence of AA (50 µg/ml) and ß-GP (10 mM). There is no apparent difference in the mineralized (dark stain) area between the transgenic and wild-type cells at either time-point. Expression of the transgene NcadC was detected at the corresponding time-points by RT-PCR of total RNA extracted from parallel cultures, with GAPDH as control for RNA integrity and abundance (lower panels). (C) Total RNA was extracted from CLVC grown for 2 weeks after confluence. The amount of mRNA for osteoblast products was determined by real-time PCR and expressed as mRNA abundance in OG2-NcadC relative to wild-type (WT) cells. Type I collagen expression levels were increased 1.8 times in transgenic animals compared with wild-type controls, whereas osteoprotegerin, osteopontin, Cbfa1 and RANK-L mRNA abundance was significantly reduced. (D) Whole-cell lysates obtained from transgenic (Tg) and wild-type (WT) CLVC grown for 3 weeks post-confluence were separated by SDS-PAGE and blotted using antibodies that recognize the intracellular tail of Xenopus N-cadherin (PEP.1) or the N-terminus of N-cadherin (N19; Santa Cruz). Two bands of 
100 and 120 kDa were detected in both cases, corresponding to endogenous N-cadherin and possibly other cross-reacting cadherins. A band of 45 kDa, corresponding to the truncated NcadC, was detected by the PEP.1 antibody only in CLVC from transgenic animals, but not from wild-type littermates (left panel). This membrane was stripped and blotted again with an anti-GAPDH antibody to control for loading.
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