Fig. 7

BMP9 and BMP2 signaling to Smad 1/5/8 depend differently on BMP type I receptors expression. Experimental conditions were as in Fig. 6, except that the transfected siRNAs were directed to ALK2, ALK3, ALK6. Scrambled siRNA served as control. a A typical blot of a signaling experiment. The experiments were conducted as in Fig. 6, and the blots were probed for pSmad1/5/8, tSmad1, and β-actin. b, c Quantification of BMP9 (b) or BMP2 (c) signaling to Smad1/5/8. Data are mean ± SEM of the pSmad1/5/8 over β-actin ratio of 4 independent experiments. The value obtained for BMP9- or BMP2-stimulated cells with siScrambled RNA was taken as 1. BMP9 signaling to pSmad1/5/8 was significantly reduced by knockdown of ALK2 or ALK3, but not ALK6. On the other hand, BMP2 signaling to pSmad1/5/8 was unaffected by knocking down either one of these type I receptors, in line with BMP2 being able to signal via multiple such receptors. d RT-qPCR quantification of ALK2, ALK3, and ALK6. Data were normalized to GAPDH as in Figs. 5 and 6. The transcript expression levels of ALK2, ALK3, and ALK6 from the Cancer Cell Line Encyclopedia [65] are given in Additional file 2: Table S1. Results are mean ± SEM of three independent experiments, each conducted in triplicate. Asterisks indicate significant differences between the pairs marked by the brackets, using one-way ANOVA and Bonferroni post hoc test for the signaling experiments (b and c; **, P < 0.002. n.s. = non-significant), and Student’s two-tailed t test for the RT-qPCR data (d; **, P < 0.005; ***, P < 5 × 10⁻⁴)