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Fig. 7. C. difficile toxin B does not inhibit internalization of ErbB2 or fluid in SK-BR-3 cells. (A) SK-BR-3 cells grown for 48 hours on poly-Lys-coated coverslips were left untreated (Con) or were treated for 2 hours with 0.5 µg/ml C. difficile toxin B (Tox), fixed, permeabilized and incubated with rhodamine phalloidin (4 U/ml). Stress fibers were seen in about half of the control cells and <1% of treated cells. (B,C) Serum-starved SK-BR-3 cells on poly-Lys-coated coverslips were left untreated (B) or were treated for 2 hours with 0.5 µg/ml C. difficile toxin B (C) before addition of FluoroRuby dextran (1 mg/ml) for 10 minutes. After fixation, surface morphology was visualized with anti-ErbB2 antibodies and green secondary antibodies. PM, plasma membrane. Scale bar: 10 µm. (D,E) Internalization of biotinylated anti-ErbB2 antibodies (D) or biotinylated BSA (E) was measured by CELISA in serum-starved SK-BR-3 cells treated with GA (D, circles), GA + C. difficile toxin B (D, squares), C. difficile toxin alone (E, squares) or left untreated (E, circles) as described in the Materials and Methods. Where appropriate, cells were pre-incubated with C. difficile toxin B (0.5 µg/ml) for 2 hours at 37°C with the addition of GA for the last 45 minutes. Values shown are the mean ± s.e.m. of three experiments.
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