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. 2016 Sep 16;6:33644. doi: 10.1038/srep33644

Figure 5. TGFBR1 expression and extraction in porcine incisor enamel organ epithelia and TR-FRET TGFBR1 kinase assay.

Figure 5

(a) TGFBR1 mRNA expression by qPCR analysis in secretory (Sec)-, transition (Tra)- and maturation (Mat)-stage ameloblasts. Each ratio was normalized to a reference gene (GAPDH) to generate relative quantification data of TGFBR1 in ameloblasts and was analysed on the basis of a mathematical model for relative quantification in the qPCR system (n = 6 ameloblasts for each stage). (b) SDS-PAGE (15% gel) stained with Simply Blue (CBB) showing both cytosol (Cyt) and membrane (Mem) fractions. Western blots showing each fraction using specific antibodies against TGFBR1 (TGFBR1-Ab) and amelogenin (Amel-Ab). TGFBR1 in the enamel organ epithelia was incubated with 100 nM of ULight-Topo IIa (Thr1342) peptide as the substrate in kinase buffer. (c) ATP titration curve: Serial dilutions of ATP ranging from 1 μM to 10 mM were added to the substrate (n = 5 wells for each concentration of sample). (d) Enzymatic time course: TGFBR1 was incubated with substrate and 5 mM ATP (blue) upon the addition of 0.5 μg of CF-hTGF-β1 (red) or 0.1 mg of P103 amelogenin-TGF-β1 complex (green) for 1.5, 3, 6 and 12 h at 37 °C (n = 5 wells for each concentration of sample). (e) Enzyme inhibition curve: Serial dilutions of SB431542 ranging from 10 nM to 1 mM were incubated with the substrate and 5 mM ATP. In all experiments, kinase reactions were terminated by the addition of 10 mM EDTA (n = 5 wells for each concentration of sample).