Fig. 4.
Fig. 4. Effects of LT or toxin B on Cdc42, Rac1, or PAK activation and actin reorganization induced by thrombin. / Prior to stimulation with thrombin for 1 minute, platelets were incubated or not with 1 μg/mL LT or toxin B for 2 hours at 37°C. (A) Pull-down of Cdc42-GTP or Rac1-GTP was performed on PBD-GST, and PAK kinase activity was tested by in vitro phosphorylation of MBP. Quantification of immunoprecipitated PAK was performed by anti-PAK1/2 immunoblot. (B,C) Platelets, preincubated with vehicle (open bars), LT (hatched bars), or toxin B (dark bars), were permeabilized and labeled with 10 μM FITC-phalloidin before analysis by (B) flow cytometry (mean ± SEM; *P < .05) or (C) confocal fluorescence microscopy (× 100). Results are representative of 3 separate experiments.

Effects of LT or toxin B on Cdc42, Rac1, or PAK activation and actin reorganization induced by thrombin.

Prior to stimulation with thrombin for 1 minute, platelets were incubated or not with 1 μg/mL LT or toxin B for 2 hours at 37°C. (A) Pull-down of Cdc42-GTP or Rac1-GTP was performed on PBD-GST, and PAK kinase activity was tested by in vitro phosphorylation of MBP. Quantification of immunoprecipitated PAK was performed by anti-PAK1/2 immunoblot. (B,C) Platelets, preincubated with vehicle (open bars), LT (hatched bars), or toxin B (dark bars), were permeabilized and labeled with 10 μM FITC-phalloidin before analysis by (B) flow cytometry (mean ± SEM; *P < .05) or (C) confocal fluorescence microscopy (× 100). Results are representative of 3 separate experiments.

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