Fig. 6
BMP9 signals to Smad 1/5/8 via ACVR2A, while BMP2 signals via ACVR2A and BMPRII. Cells were transfected with siRNA to ACVR2A, BMPRII, or siScrambled RNA (control). After 24 h, they were taken for signaling studies (a–d) or RT-qPCR determination of the mRNA levels of ACVR2A (Fig. 4 c) or BMPRII (e). a, c Representative signaling blots. Cells were starved (2 h, 1% serum), stimulated (30 min, 37 °C) or not (control) with 4 nM BMP9 (a) or 10 nM BMP2 (c), lysed, and subjected to SDS-PAGE followed by immunoblotting for pSmad1/5/8, tSmad1, and β-actin. b, d Quantification of BMP9 (b) and BMP2 (d) signaling to Smad1/5/8. Bands were visualized by ECL and quantified by densitometry. Data are mean ± SEM of the pSmad1/5/8 over β-actin ratio of 4 independent experiments. The value obtained for BMP-stimulated cells transfected with siScrambled RNA was taken as 1. pSmad1/5/8 formation in response to BMP9 (a, b) was markedly abrogated by siACVR2A (and siACVR2B; Additional file 1: Fig. S7c, d), but was unaffected by BMPRII siRNA. On the other hand, BMP2 signaling (c, d) to pSmad1/5/8 was reduced ~2-fold by siRNA to ACVR2A or BMPRII, but not by siRNA to ACVR2B (Fig. S7e, f). e RT-qPCR quantification of BMPRII (ACVR2A is shown in Fig. 5 c). Data were normalized to GAPDH, taking the BMPRII mRNA level in siScrambled cells as 1. The transcript expression level of BMPRII from the Cancer Cell Line Encyclopedia [65] is 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 bracketed pairs (one-way ANOVA and Bonferroni post hoc test for signaling studies; b, d) or Student’s two-tailed t test for RT-qPCR (e). **, P < 0.001; ***, P < 10−4. n.s. = non-significant (P > 0.5)