Figure 6. The FBG domain of TNX devoid... | Rockefeller University Press

Figure 6.

$Figure 6. The FBG domain of TNX devoid of TGF-β activity retains the ability to activate cell-secreted latent TGF-β1. (A) The FBG domain was stripped of TGF-β1–associated activity under alkaline conditions. (top) ELISA quantitation of total active human TGF-β1 was performed with 2.5 µg protein from untreated FBG domain (input fraction) or from unbound and bound fractions of purified FBG domain under alkaline conditions. (bottom) Immunoblot analyses of TGF-β1 and the FBG domain were performed in parallel on the same fractions (2.5 µg protein). (B) Western blot analysis of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h with the different fractions (50 µg/ml) containing the FBG domain stripped of TGF-β1–associated activity (bound fraction) or unbound (flow through) and wash fractions as described in A. As a control, untreated or alkaline-treated BSA was used instead of FBG domain. Ratio of phospho-Smad2 to actin levels is indicated below. (C) Firefly luciferase activity in NMuMG cells transiently transfected with the Smad-responsive (CAGA)9-Luc reporter construct. The cells were incubated for 24 h with either recombinant TGF-β1 (5 ng/ml) or the different fractions (50 µg/ml) containing the FBG domain subjected or not subjected to alkaline treatment described in A. N-C, noncoated. (D, top) SDS-PAGE analysis of purified FBG domain produced in mammalian cells (FBG) and in E. coli (FBG*). MM, molecular mass markers. (bottom) Immunoblot analysis of the levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with purified FBG domain (111 nM each). (E) Immunoblot analysis of phospho-Smad2 in NMuMG cells cultured for 3 h with 5 ng/ml TGF-β1, soluble purified FBG domain (111 nM) produced in mammalian cells (FBG) or in E. coli (FBG*), or the corresponding vehicle (PBS). Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (F) Firefly luciferase activity of MCF10A-TRE-Luc cells incubated for 24 h as described in E. (G) F-actin direct fluorescence (top) and indirect immunofluorescence staining of E-cadherin (bottom) performed in NMuMG cells cultured for 48 h with soluble FBG domains produced in eukaryotic (FBG) or prokaryotic (FBG*) system (111 nM). Bars, 15 µm. *, P < 0.05 versus control condition. Error bars are means ± SD. WB, Western blot.$

The FBG domain of TNX devoid of TGF-β activity retains the ability to activate cell-secreted latent TGF-β1. (A) The FBG domain was stripped of TGF-β1–associated activity under alkaline conditions. (top) ELISA quantitation of total active human TGF-β1 was performed with 2.5 µg protein from untreated FBG domain (input fraction) or from unbound and bound fractions of purified FBG domain under alkaline conditions. (bottom) Immunoblot analyses of TGF-β1 and the FBG domain were performed in parallel on the same fractions (2.5 µg protein). (B) Western blot analysis of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h with the different fractions (50 µg/ml) containing the FBG domain stripped of TGF-β1–associated activity (bound fraction) or unbound (flow through) and wash fractions as described in A. As a control, untreated or alkaline-treated BSA was used instead of FBG domain. Ratio of phospho-Smad2 to actin levels is indicated below. (C) Firefly luciferase activity in NMuMG cells transiently transfected with the Smad-responsive (CAGA)₉-Luc reporter construct. The cells were incubated for 24 h with either recombinant TGF-β1 (5 ng/ml) or the different fractions (50 µg/ml) containing the FBG domain subjected or not subjected to alkaline treatment described in A. N-C, noncoated. (D, top) SDS-PAGE analysis of purified FBG domain produced in mammalian cells (FBG) and in E. coli (FBG*). MM, molecular mass markers. (bottom) Immunoblot analysis of the levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with purified FBG domain (111 nM each). (E) Immunoblot analysis of phospho-Smad2 in NMuMG cells cultured for 3 h with 5 ng/ml TGF-β1, soluble purified FBG domain (111 nM) produced in mammalian cells (FBG) or in E. coli (FBG*), or the corresponding vehicle (PBS). Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (F) Firefly luciferase activity of MCF10A-TRE-Luc cells incubated for 24 h as described in E. (G) F-actin direct fluorescence (top) and indirect immunofluorescence staining of E-cadherin (bottom) performed in NMuMG cells cultured for 48 h with soluble FBG domains produced in eukaryotic (FBG) or prokaryotic (FBG*) system (111 nM). Bars, 15 µm. *, P < 0.05 versus control condition. Error bars are means ± SD. WB, Western blot.