Fig. 4: T3SS-delivered RAS inhibitors downregulate RAS signaling.
From: Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol
a Western blot analysis of uninfected HCT116 cells (Uninf) and HCT116 cells infected with Salmonella ASB2519 (1 h, MOI of 100) delivering a FLAG-tagged SptP120-control (Ctrl) DARPin and the SptP120-anti-RAS inhibitory DARPins K27 and K55 or the RAS blocking monobody NS1. Anti-FLAG detection served as the transfer control and total anti-ERK1/2, AKT and Actin as loading controls. Numbers below the pERK1/2 and pAKT blots detected with anti-pERK1/2 (Thr202/Tyr204) and anti-pAKT (Ser473) antibodies represent the quantified fraction relative to the SptP120-control DARPin (Ctrl), normalized to total ERK1/2 and AKT, respectively. Uncropped blots can be found in Supplementary Fig. 10. b Flow cytometric measurements of ERK1/2 phosphorylation in HCT116 cells upon FLAG-positive delivery of the indicated SptP120-anti-RAS binders. Data were analyzed 10 min post-infection in the presence of bortezomib (BZB; 50 nM). Pink lines indicate baseline ERK1/2 phosphorylation levels upon delivery of the SptP120-control DARPin. Relative median fluorescence intensities (MFI) of ERK1/2 (c) and GSK3β (d) phosphorylation compared to the SptP120-control DARPin (Ctrl) treated cells and using the same experimental setup as in b. e Flow cytometric analysis of ERK1/2 phosphorylation in HeLa cells upon FLAG-positive delivery of the indicated SptP120-anti-RAS binders and a control DARPin. Data were analyzed 10 min post-infection in the presence of bortezomib (BZB) without (empty) or with EGF (20 ng per mL) stimulation (filled). Relative median fluorescence intensities (MFI) of ERK1/2 (f) and GSK3β (g) phosphorylation compared to the SptP120-control DARPin (Ctrl) treated cells with EGF and using the same experimental setup as in e. Data represent the mean ± SEM of six (c, d) or three (f, g) biological replicates. Individual data points are shown. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns not significant; only relevant comparisons are indicated for (f) and (g)).
