Fig. 3

Detection of TGF-β activity in the G2 extract. A Flow chart showing the procedures used for the primary extracts of proteins from bone powder. Sup: supernatant, Ppt: precipitate, RIM: residual insoluble materials. B Rat bone powder extracts analysed by SDS–PAGE stained with Bio-Safe Coomassie Brilliant Blue. M: SeeBlue® Plus2 Pre-Stained Standard. C ALP-inducing activity of HPDL cells exposed to the G2 extract (250 μg/mL) and CF-hTGF-β1 (1 ng/mL) without (−) or with (+) SB431542. Data in box indicate the 25–75% tiles of the interquartile range (IQR), and the end of whiskers indicates maximum and minimum values, respectively. The median is represented as line located in the middle of box (*p < 0.05, Mann–Whitney U test). Control: ALP-inducing activity of HPDL cells cultured in the absence of the G2 extract or CF-hTGF-β1. D Dual-luciferase reporter-gene assay for the G2 extract. Firefly luciferase activity exposed to the G2 extract (250 μg/mL) and CF-hTGF-β1 (1 ng/mL) without (−) or with (+) SB431542. Firefly luciferase activity was standardized based on Renilla luciferase activity, which is an internal standard. Data in box indicate the 25–75% tiles of IQR, and the end of whiskers indicates maximum and minimum values, respectively. The median is represented as line located in the middle of box (*p < 0.05, Mann–Whitney U test). Control: dual-luciferase reporter gene assay of mHAT9d cells cultured in the absence of the G2 extract or CF-hTGF-β1