Fig. 4: Quantitative western blots and ELISA show that fortilin blocks TGF-β1-induced phosphorylation of Smad3.
From: Fortilin interacts with TGF-β1 and prevents TGF-β receptor activation
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); α-TGF-β1 mAb, neutralizing α-TGF-β1 monoclonal antibody (1.25 µg/mL or 8.33 nM); IB, immunoblot; α-P-Smad3, anti-phosphorylated Smad3 antibody; α-Smad3, anti-Smad3 antibody; TCE, 2,2,2-trichloroethanol; A.U., arbitrary unit. a Western blot analysis using α-P-Smad3 and α-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-β1 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-β1 from phosphorylating Smad3.
