Figure 4

α-def-6 inhibits intracellular Rac1 glucosylation by TcdA, TcdB and the combination of both toxins. (A) Vero cells were treated with TcdA (20 pM), TcdB (10 pM) or the combination of both toxins with or without the addition of α-def-6 (concentrations as indicated) for 5 h (TcdB) or 4 h (TcdA and TcdA + TcdB), respectively. Then, the cells were harvested, lysed and subjected to SDS-PAGE followed by Western blotting. Non-glucosylated Rac1 was detected and signals were normalized to GAPDH as loading control. Representative Western blots are depicted. Relative signal intensities are given as mean ± SD of at least three biological replicates (n ≥ 3). Significance was tested with one-way ANOVA combined with Dunnett’s multiple comparison test (* p < 0.05, *** p < 0.001). (B) Vero cells were treated as in (A). α-def-6 (6 µM) was used alone for control. After 4 h, cells were fixed and permeabilized. Non-glucosylated Rac1 (red) was stained with an anti-Rac1-antibody in combination with a secondary Alexa Fluor₆₃₃-conjugated antibody. Actin (green) was stained with phalloidin-FITC and nuclei (blue) via Hoechst33342. Representative images are shown (n = 2). Scale bars correspond to 50 µm.