Figure 5: Anti-Ras DARPins engage Ras within the intracellular environment.
From: Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange
In a mammalian two-hybrid assay, interaction of an H-Ras-DNA-binding domain fusion and a DARPin-VP16 transactivator fusion, following transient transfection of expression plasmids, enables binding to a Gal4 binding site and activation of a firefly luciferase reporter in the cell line CHO-Luc15 (a). Luciferase signals resulting from co-expression of various DARPin and H-Ras fusion pairs (b) demonstrate that the majority of anti-Ras DARPins interact with H-Ras within the reporter cell, regardless of their inhibitory mechanism or preference for inactive or active Ras. Error bars represent the mean±s.d. (n=3). FLAG-tagged DARPin expression in an inducible HCT116 cell line followed by FLAG-tag immunoprecipitation demonstrates that DARPin K27, but not a control DARPin, can bind and capture endogenous Ras within a tumour cell line (c).
