Figure 7: Anti-Ras DARPin K27 and K55 expression in HEK293 and HCT116 cells causes inhibition of ERK phosphorylation.
From: Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange
HEK293 cells were co-transfected with plasmids encoding (i) K-Ras and (ii) DARPins to study changes in ERK phosphorylation (a). Thirty hours post-transfection, HEK293 cells were lysed for Western blot analysis with the antibodies indicated. For flow cytometric measurement of ERK phosphorylation, HCT116 colorectal cancer cells expressing K27, K55 or control DARPin E3_5 were stimulated with EGF for 0, 10, 15 or 20 min before analysis. For each condition histograms of phosphorylated ERK (pERK) are shown and median fluorescence intensity (MFI) is plotted. HCT116 cells were collected either 24 h post transfection with plasmids encoding the DARPins, and subsequently gated on cells expressing high levels of FLAG-tagged DARPins (b) or HCT116 cell lines with lentivirally transduced DARPin gene under a doxycycline-inducible promoter were used and assayed after 48 h of culture with or without doxycycline (c).
