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Fig. 5. Effect of BAPTA on CAL-stimulated gliding and secretion. (A) Quantification of the length of SAG1-labeled trails deposited by wild-type parasites. Simultaneous BAPTA and CAL treatment did not stimulate increased trail length, unlike CAL treatment alone. 1 µM CD was used as a negative control for gliding. Bars show average trail length in parasite body lengths (mean±s.e.m.). *A significant difference when compared to control trail lengths; P<0.05, two-tailed Student's t test. (B) Western blotting of parasite supernatant proteins showed that CAL-stimulated secretion of MIC2 was blocked by BAPTA. Stimulation with 1% ethanol was used as a positive control for secretion. Supernatants were compared to diluted lysates of cell standards (% pellets). Blots were probed with monoclonal antibody 6D10 (cellular MIC2 (cMIC2) and secreted MIC2 (sMIC2). (C) Phosphorimager analysis of western blots demonstrated that the amount of MIC2 secretion no longer increased after BAPTA treatment, even with increasing concentrations of CAL. Bars represent the average of three experiments (mean±s.e.m.). (D) Caffeine treatment did not increase length of trails formed by parasites during a gliding assay. 1 µM CD was used as a negative control for gliding. Bars show average trail length in parasite body lengths (mean±s.e.m.).
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