Fig. 1. Effect of PTH on Runx-2 protein levels in COBs from Fgf2+/+ and Fgf2−/− mice.
Expression levels of Runx-2 in COBs were analyzed by Western blotting as described under methods. A, COBs from Fgf2+/+ and Fgf2−/− mice were treated with PTH (10−8 M to10−10 M) for 24 h. Proteins (5 µg) from each sample were subjected to SDS–PAGE, transferred to PVDF membrane and probed with a polyclonal anti-Runx-2 antibody. Filters were stripped and reprobed with a monoclonal anti-α-tubulin antibody to show equal amount of loading. B, COBs from Fgf2+/+ and Fgf2−/− were treated with PTH 10−9 M from 15 min to 24 h. Proteins (5 µg) from each sample were subjected to SDS–PAGE, transferred to PVDF membrane and probed with a polyclonal anti-Runx-2 antibody. Filters were stripped and reprobed with a monoclonal anti-α-tubulin antibody, to show equal amount of loading. C, Statistical analysis from a pool of four independent experiments showed that 24 h of PTH treatment increased Runx-2 protein only in Fgf2 +/+ COBs (Mean± SD. *p <0.05). D, Effect of PTH and FGF-2 on Runx-2 labeling pattern in COBs from Fgf2+/+ and Fgf2−/− mice. COBs from Fgf2+/+ and Fgf2−/− were treated with PTH 10−9 M or FGF-2 10−9 M for 24h. Localization of Runx-2 was analyzed by fluorescent microscopy using a polyclonal anti-Runx-2 antibody as described under methods. Inserts clearly demonstrate the perinuclear and nuclear accumulation of Runx-2 after PTH treatment only in Fgf2+/+ COBs (green: FITC staining). Bar, 50 µm.