Fig. 2.

Exogenous heparin inhibits BMP6-mediated signaling. a BMP6 induces Smad1/5/8 phosphorylation. C2C12-BRE-Luc cells were stimulated with BMP6 (50 ng/ml) for indicated time points. b Cells were stimulated with BMP6 for 30 min or BMP6 and heparin (10 μg/ml) for various periods of time (30 min–9 h). c C2C12-BRE-Luc cells were stimulated with BMP6 alone or in combination with heparin, chondroitin sulphate and de-O- and de-N-sulphated heparin for 17 h. BMP6-mediated signalling is inhibited by heparinase III and chlorate treatment. d, e Heparinase III pretreatment interferes with BMP6-mediated signalling. C2C12-BRE-Luc cells were incubated in the presence or absence of heparinase III (25 mIU/ml) for three hours and then stimulated with BMP6 (50 ng/ml) for 30 min (d) or 10 ng/ml for 17 h (e). f, g Chlorate treatment inhibits BMP6-mediated signalling. C2C12-BRE-Luc were incubated with or without sodium chlorate (50 mM) for four days and then stimulated with BMP6 (50 ng/m) for 30 min (f) or 10 ng/ml for 17 h (g). a, b Cells were lysed and 45-μg aliquots were subjected to immunoblot analysis with an anti-phospho-Smad1/5/8 antibody as described in Materials and methods. β-Actin levels are shown as a loading control. Total Smad 1/5/8 levels showed no difference between samples. c, f, g The cells were lysed and the luciferase activity measured using Victor Wallack luminometer as described in Materials and methods. °P < 0.05, comparison between BMP6 with heparin and BMP6 alone; *P < 0.05, comparison between BMP6 with and without heparinase III/chlorate; ** P < 0.05, comparison between BMP6 without and with heparin; #P < 0.05, comparison between BMP6 without and with heparin, all with heparinase III