Fig 1: The luciferase assay shows that fortilin blocks TGF-ß1-induced activation of the Smad2/3 binding element.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; a-TGF-ß1 mAb, neutralizing anti-TGF-ß1 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-ß1 from activating the SBE in HEK293SBE-Luc cells; 1 nM TGF-ß1 and 1 nM strep-tag-fortilin were used. N = 4. b Fortilin, but not luciferase control protein, dose-dependently blocked TGF-ß1-induced SBE activation in the MFB-F11SBE-SEAP cells; 156 pM TGF-ß1 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-ß1-induced SBE activation. N = 6.
Fig 2: Quantitative western blots and ELISA show that fortilin blocks TGF-ß1-induced phosphorylation of Smad3.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; strep-tag-fortilin, recombinant fortilin with strep-tag at its N-terminus (19.5 nM); strep-tag-luciferase, recombinant luciferase with strep-tag at its N-terminus, used as the control (19.5 nM); a-TGF-ß1 mAb, neutralizing a-TGF-ß1 monoclonal antibody (1.25 µg/mL or 8.33 nM); IB, immunoblot; a-P-Smad3, anti-phosphorylated Smad3 antibody; a-Smad3, anti-Smad3 antibody; TCE, 2,2,2-trichloroethanol; A.U., arbitrary unit. a Western blot analysis using a-P-Smad3 and a-Smad3 and total protein visualization by TCE. b, c Quantification of P-Smad3, normalized to total proteins (b) and total Smad3 (c), showing that fortilin, but not luciferase control protein, prevented TGF-ß1 from phosphorylating Smad3. d ELISA of P-Smad3 on the lysates from MFB-F11SBE-SEAP cells also showed that fortilin, but not luciferase control protein, prevented TGF-ß1 from phosphorylating Smad3.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human LAP (TGF-beta 1) Protein, CF