Fig. 4. Depletion of endogenous FGF-2 expression by siRNA in COBs from Fgf2+/+ mice.
A, Cells were transfected with FGF-2 siRNA or control siRNA and the level of FGF-2 protein was analyzed by Western blotting as described under methods. Proteins (5 µg) from each sample were subjected to SDS–PAGE, transferred to PVDF membrane and probed with a polyclonal rabbit anti-FGF-2 antibody. Filters were stripped and reprobed with a monoclonal anti-α-tubulin antibody to show equal amount of loading. B, After transfection, as above described, cells were serum deprived for 24h and treated with PTH 10−9 M or vehicle from another 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 or a polyclonal anti-phospho-CREBs antibody; then 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 three different experiments showed that PTH increases Runx-2 and phospho-CREBs levels only in presence of FGF-2 (Mean± SD *p <0.05).