Figure 4.

Activation of p-SMAD-1/5/9 by small molecules. A, immunoblotting of whole cell protein lysates from BRE-Luc cells treated with either 2 ng/ml rhBMP4, 0.04% DMSO, or the top 12 BMP candidate agonists at 10 μm for 1 h in BRE-Luc cells. Membranes were probed with anti-p-SMAD-1/5/9 and anti-total-SMAD-1. β-Actin served as an additional loading control. B, quantitation of p-SMAD-1/5/9 protein levels as determined by densitometry of three independently derived Western blots. The p-SMAD-1/5/9 levels were normalized to total-SMAD-1. Final data are expressed as -fold change relative to the mean negative control signal, with error bars representing 1 S.D. (*, p < 0.05; **, p < 0.01; ****, p < 0.0001 relative to control). ANOVA, Dunnett's post hoc multiple comparisons test was used to determine significance. C, immunoblotting of lysates from serum-starved PRECs treated for 24 h with increasing concentrations of sb4 in serum-free medium. Total SMAD-1 and β-actin serve as loading controls. D, quantitation of p-SMAD-1/5/9 levels are shown. Statistical analyses of replicates were done as in B.