Fig. 2
Binding of Hp-TGM to the TGF-β Receptors. a MFB-F11 TGF-β-responsive bioassay for activity following 24 h of culture at 37 °C, comparing Hp-TGM to hTGF-β1 and the complex HES mixture by protein concentration. MFB-F11 cells are transfected with a Smad-responsive plasmid construct in which TGF-β binding leads, through Smad phosphorylation and nuclear translocation, to expression of alkaline phosphatase, which is measured following the addition of p-nitrophenyl phosphate. Data shown are representative of > 3 independent experiments, and represent mean ± SEM from duplicate wells. b–e Surface plasmon resonance analysis of Hp-TGM and hTGF-β3 binding to hTGF-β receptors. Streptavidin-coated biosensor chips were loaded with biotinylated Hp-TGM or hTGF-β3 and two-fold dilutions of the ectodomain of hTβRII (from 13 µM downwards) b, c and of the ectodomain of hTβRI (from 4 µM downwards) d, e were passed over the Hp-TGM or hTGF-β3 surface, respectively. Data shown are from one of two similar experiments. f, g Surface plasmon resonance analysis of Hp-TGM and hTGF-β3 binding to TβRI in the presence of near-saturating TβRII (2 μM), showing independent binding by Hp-TGM to both receptors, but binding of hTGF-β3 to TβRI dependent on the presence of TβRII
