Fig. 5. Effect of the NO-cGMP-PKG pathway on Cbfa-1 regulation in MC3T3-E1 cells. (A) Immunoblot analysis of Cbfa-1 over a time course of MC3T3-E1 differentiation (n=3). (B) Immunoblot analysis of Cbfa-1 over a time course of MC3T3-E1 differentiation in the presence of DEA-NO or 8Br-cGMP (n=3). (C) Immunocytochemistry analysis of Cbfa-1 (red) at day 7 of MC3T3-E1 differentiation. Cells were induced to differentiate (ii), or not (i), or induced to differentiate in the presence of 200 µM 1400W (iii). Nuclei were visualized with Hoechst (blue) (n=3). Bars, 25 µm. (D) Cbfa-1 expression from nuclear extracts and cytosolic extracts of MC3T3-E1 after 7 and 14 days of differentiation. PARP was detected as a control for nuclear integrity (n=3). (E) Recombinant Cbfa-1 (2 µg) was incubated with 100 units of PKG or 100 units of denatured PKG (bPKG) in phosphorylation buffer (see Materials and Methods), containing 1 µCi of [³²P]-ATP, in the presence or absence of 100 µM cGMP. Samples were electrophoresed and phosphorylation was visualized by autoradiography (n=2).