Fig. 3: The luciferase assay shows that fortilin blocks TGF-β1-induced activation of the Smad2/3 binding element.
From: Fortilin interacts with TGF-β1 and prevents TGF-β receptor activation
strep-tag-fortilin, recombinant fortilin with strep-tag at its N-terminus; strep-tag-luciferase, recombinant luciferase with strep-tag at its N-terminus, used as the control; α-TGF-β1 mAb, neutralizing anti-TGF-β1 monoclonal antibody (1.25 µg/mL or 8.33 nM); HEK, human embryonic kidney cells; SBE-Luc, a vector containing the Smad2/3 binding element fused to the luciferase cDNA; A.U., arbitrary unit; SBE-SEAP, a vector containing the Smad2/3 binding element fused to the secreted embryonic alkaline phosphatase cDNA; MFB-F11SBE-SEAP cells, immortalized mouse embryonic fibroblasts from Tgfb1−/− mice that stably harbor the SBE-SEAP construct; data points, means ± SD; statistical analyses performed using ANOVA with Fisher’s multiple comparison; N, the number of biological replicates; NS, not statistically significant; ****P < 0.001. a Fortilin prevented TGF-β1 from activating the SBE in HEK293SBE-Luc cells; 1 nM TGF-β1 and 1 nM strep-tag-fortilin were used. N = 4. b Fortilin, but not luciferase control protein, dose-dependently blocked TGF-β1-induced SBE activation in the MFB-F11SBE-SEAP cells; 156 pM TGF-β1 was used to stimulate the cells. Moreover, 19.5 (low dose, +) and 195 (high dose, ++) nM strep-tag-fortilin or strep-tag-luciferase were used to block TGF-β1-induced SBE activation. N = 6.
