Fig. 8

ALK4 and type I BMP receptors compete for signaling to distinct Smad pathways via ACVR2. U2OS cells were transfected with empty vector (control) or one of the type I receptors (HA-tagged ALK2, ALK3, ALK6, or ALK4). At 24 h post-transfection, they were starved (2 h, 1% serum) and stimulated (30 min, 37 °C) where indicated with 4 nM ActA or 4 nM BMP9. The cells were then subjected to lysis, SDS-PAGE and immunoblotting, probing for pSmad2/3, tSmad2/3 (for ActA signaling), pSmad1/5/8, tSmad1 (for BMP9 signaling), as well as for HA tag (using HA-7 αHA) and β-actin. a, b Representative blots of the effects of overexpression of ALK2, ALK3, or ALK6 on ActA signaling to pSmad2/3. c A representative blot showing the effect of ALK4 overexpression on BMP9 signaling to pSmad1/5/8. The expression of the various HA-tagged receptors was probed by blotting for the HA tag (a–c). d, e Quantification of ALK2/3/6 effects on ActA-mediated pSmad2/3 formation (d) and of ALK4 on BMP9-mediated pSmad1/5/8 formation (e). Data are mean ± SEM of the relevant pSmads over β-actin ratio of 4 independent experiments. The value obtained for control cells stimulated with ActA (d) or with BMP9 (e) was taken as 1. Asterisks indicate significant differences between the pairs marked by the brackets, using one-way ANOVA and Bonferroni post hoc test (*, P < 0.002; **, P <0.0001)