Fig. 1
Ligands induced less BMP and TGF-β signaling in iMSCACVR2B-Fc than in iMSCControl. A Schematic linear maps of the plasmids used in the study. ITR, inverted terminal repeat; CAG, EF1a, CMV: promoter; IRES, internal ribosome entry site. B Representative images of stably expressing cells after puromycin selection for 7 d. Scale bar, 300 µm. C The relative mRNA levels of ACVR2B in iMSCACVR2B-Fc and control cells based on qPCR analysis. D Protein levels of ACVR2B in iMSCACVR2B-Fc and the control cells based on western blotting. β-actin acts as an internal reference. All full-length blots are presented in Additional file 2: Fig. 1D. E The concentration of His-tag proteins in the cell supernatants at two-time points based on ELISA. F Activin-A, BMP-7, and BMP-9 induced less BRE-Luc activity in iMSCACVR2B-Fc than in iMSCControl. G Representative image of western blotting analysis. BMP-9 induced less phosphorylation of SMAD1/5/8 (p-SMAD1/5/8) in iMSCACVR2B-Fc than in iMSCControl. All full-length blots are presented in Additional file 2: Fig. 1G. H Quantification of relative p-SMAD1/5/8 levels normalized to β-actin. I TGF-β3 induced less CAGA-Luc activity in iMSCACVR2B-Fc than in iMSCControl. J Representative image of western blotting analysis. Activin-A and TGF-β3 induced less phosphorylation of SMAD2/3 (p-SMAD2/3) in iMSCACVR2B-Fc than in iMSCControl. All full-length blots are presented in Additional file 2: Fig. 1 J. K Quantification of relative p-SMAD2/3 levels, normalized to β-actin. L Representative image of western blotting analysis. FK506 initiated increased Activin-A-dependent activation of BMP signaling in iMSCControl, compared with that in iMSCACVR2B-Fc. All full-length blots are presented in Additional file 2: Fig. 1L. M ACVR2B-Fc suppressed the Activin-A-dependent activation of TGF-β signaling and inhibited the enhanced activation of TGF-β signaling induced by FK506. All full-length blots are presented in Additional file 2: Fig. 1 M. Activin-A, 100 ng/ml; BMP-7, 100 ng/ml; BMP-9, 10 ng/ml; TGF-β1 & TGF-β3, 10 ng/ml; FK506, 1 µM. Results represent the mean ± SEM. n.s., no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001 by unpaired t-test for qPCR and two-way ANOVA with Å Ãdák's multiple comparisons test for others