Fig 2: Expression of Erk1/2, p-Erk1/2 and Ras in Tregs. Tregs were cultured with anti-CD3 antibody, anti-CD3 + anti-CD28 antibody or anti-CD3 + anti-ICOS antibody for 4 days. Western blot was used to evaluate the expression of Erk1/2, p-Erk1/2 and Ras. All of the experiments were performed in triplicate, ** P<0.01.

Fig 3: The glucosyltransferase domain of TpeL is transported effectively via PA into Panc-1 but not into Capan-2 cells(A) LFN-TpeL inhibits the Ras pathway in Panc-1 cells. Cells were intoxicated for 6 h with the indicated concentrations of LFN-TpeL and a fixed concentration of PA (10 nM). (B) The Western blot is a representative of three independent experiments. LFN-TpeL inhibits proliferation of Panc-1 cells. Cells were intoxicated as in A. Cell proliferation was measured by the incorporation of BrdU in intoxicated cells compared to an untreated control. The figure shows the result of three independent experiments. (C) LFN-TpeL is not effective in Capan-2 cells. Cells were intoxicated for 4 h at the indicated concentrations of LFN-TpeL and a fixed concentration of PA (10 nM). The Ras downstream signaling was not altered after intoxication. Data shown are a representative of three independent experiments.

Fig 4: Inactivation of (H/K/N)Ras in TcdA-treated Vero cells. Vero cells were exposed to native TcdA-10463 and TcdB-10463 for 16 h. Cells were lysed and objected to effector pull-down assay exploiting the Ras-binding domain of Raf kinase. The cellular levels of GTP-loaded (H/K/N)Ras, total H-Ras, pT202/pY204-p44/42-MAP kinase (pT202/pY204-ERK1/2), and total p44/42-MAP kinase (ERK1/2) were analyzed by immunoblot analysis using the indicated antibodies.

Fig 5: RAS‐driven EV internalization involves surface fibronectin‐HSPG interactions. (a) Confocal images (field of a 63× objective lens) show the decreased EV uptake following heparin co‐treatment of RAS3 cells. (b) Proteomes of IEC18 EVs and RAS3 EVs (Chennakrishnaiah et al., 2020) (three biological replicates) highlight the expression of HSPGs and HSPG binding proteins that could play a role in EV‐cellular interactions (see the GO analyses of proteomes in Figure S1). (c) High salt washing by 1 M KCl results in a complete disappearance of FN without affecting the levels of intravesicular marker ALIX. (d) Confocal images (field of a 40× objective lens) show the decreased uptake of KCl‐treated EVs by RAS3 cells. Internalized PKH26‐labeled EVs in confocal images were quantified by the total number of PKH26‐positive particle spots per cell (likely representative of clusters containing multiple EVs). Counting was conducted in 7 or 8 randomly taken images of individual cells. (e) Western blotting shows that Hrase I/III (heparinase) treatment removed the FN from EVs without affecting CD9 levels. (f) Confocal images (field of a 40× objective lens) show the decreased uptake of Hrase I/III‐pre‐treated EVs by RAS3 cells. P values (here and elsewhere: **** < 0.0001; *** < 0.001; ** < 0.01)