Figure 1.
Blockade of TGF-β signaling with synthetic Fabs identified by phage display screening.
a) The extracellular domain of TGFBR2 was inserted between an IL2 signal sequence (N-terminus) and the human Fc IgG1 domain (C-terminus). b) Expression and capture of the Fc and Fc-ECD proteins from transfected HEK293F cell conditioned media (CM) using Protein A Sepharose beads and immunoblot (IB) with anti-human Fc (mock represents no transfection, positions of molecular mass markers indicated on the left). c) Affinity constants (KD) represent the interactions between unique Fabs and the TGFBR2-Fc protein, as measured by SPR. d) A Dual luciferase assay was used to measure effects of Fab treatments on Smad2/3 activity in HEK293T cells treated with TGFβ1 (10 ng/ml) for the final 10 hours of a 48 hour period post transfection (* p < 0.05, *** p < 0.001). e) Graph depicts densitometry analysis of immunoblot results for the relative SMAD2/3 phosphorylation levels in TGF-β-treated MDA-MB-231 cells with or without pretreatment with the indicated Fab’s (500 nM).